# Patent

## Title
Piscine reovirus immunogenic compositions

## Abstract

The invention is directed to immunogenic compositions and methods for inducing an immune response against Piscine reoviruses in an animal. In another aspect, the invention relates to antibodies that bind Piscine reovirus poplypeptides. In yet another aspect, the invention relates to methods for preventing, or reducing PRV infection in an animal.

## Background
This application is a U.S. National Phase Application under 35 U.S.C. 371 of International Patent Application No. PCT/US10/51346 filed Oct. 4, 2010, which claims the benefit of and priority to U.S. provisional patent application Ser. No. 61/248,058 filed Oct. 2, 2009, U.S. provisional patent application Ser. No. 61/325,047 filed Apr. 16, 2010, and U.S. provisional patent application Ser. No. 61/380,594 filed Sep. 7, 2010, the disclosures of all of which are hereby incorporated by reference in their entireties for all purposes.


This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The patent and scientific literature referred to herein establishes knowledge that is available to those skilled in the art. The issued patents, applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail.
BACKGROUND
Fish are an increasingly important source of food and income; global annual consumption projected to rise from 110 million tons in 2010 to more than 200 million tons in 2030. Whereas rates of wild fish capture are flat or declining due to overfishing and loss of habitat, the global mariculture harvest is growing at a rate in excess of 8% per annum. However, the emergence of infectious diseases in aquaculture threatens production and may also impact wild fish populations. Atlantic salmon (Salmo salar L.) are amongst the most popular of farmed fish, accounting for annual production of more than 1 million tons. Atlantic salmon mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens, or fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999 (Kongtorp et al., J Fish Dis 27, 351-358 (2004)), HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom (Ferguson et al., J Fish Dis 28, 119-123 (2005)). Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful.
HSMI is transmissible but the causal agent has not been previously isolated. HSMI is an important disease that threatens aquaculture. There is a need for immunogenic compositions and vaccines suitable for preventing and containing PRV infection and for treating animals having HSMI. This invention addresses these needs.
SUMMARY OF THE INVENTION
The invention relates to Piscine reovirus (PRV), a novel orthoreovirus-like virus associated with Salmon HSMI, and isolated nucleic acids sequences and peptides thereof. The invention is also related to antibodies against antigens derived from PRV and method for generating such antibodies. The invention is also related to immunogenic compositions for inducing an immune response against PRV in an animal.
In one aspect, the invention provides a PRV immunogenic composition comprising a PRV nucleic acid. In one embodiment, the PRV nucleic acid is a nucleic acid sequence of any of SEQ ID NOs: 1-10. In another embodiment, the PRV nucleic acid comprises least 24 consecutive nucleic acids of any of SEQ ID NOs: 1-10. In still another embodiment, the PRV nucleic acid is substantially identical to the nucleic acid sequence of any of SEQ ID NOs: 1-10. In still a further embodiment, the PRV nucleic acid is a variant of any of SEQ ID NOs: 1-10 having at least about 85% identity to SEQ ID NOs: 1-10. In one embodiment, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any one of SEQ ID NOs: 1-10.
In yet another aspect, the invention provides a PRV immunogenic composition comprising a PRV polypeptide. In one embodiment, the PRV polypeptide is a polypeptide encoded by any of SEQ ID NOs: 1-10. In yet another embodiment, the PRV polypeptide is a polypeptide encoded by a nucleic acid comprising least 24 consecutive nucleic acids of any of SEQ ID NOs: 1-10. In still a further embodiment, the PRV polypeptide is a polypeptide encoded by a nucleic acid that is substantially identical to the nucleic acid sequence of any of SEQ ID NOs: 1-10. In still a further embodiment, the PRV polypeptide polypeptide is a polypeptide encoded by a nucleic acid that is a variant of any of SEQ ID NOs: 1-10 having at least about 85% identity to SEQ ID NOs: 1-10. In still a further embodiment, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any one of SEQ ID NOs: 1-10. In yet another embodiment, the PRV polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NOs: 29-40. In yet another embodiment, the PRV polypeptide is a polypeptide comprising least 8 consecutive amino acids of any of SEQ ID NOs: 29-40. In still a further embodiment, the PRV polypeptide is substantially identical to the amino acid sequence of any of SEQ ID NOs: 29-40. In still another embodiment, the PRV polypeptide is a variant of any of SEQ ID NOs: 29-40 and having at least about 85% identity to SEQ ID NOs: 29-40. In still a further embodiment, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any one of SEQ ID NOs: 29-40.
In another aspect, the invention provides an antibody that binds a PRV or a PRV polypeptide and inhibits, neutralizes or reduces the function or activity of the PRV or PRV polypeptide. In one embodiment, the antibody is a polyclonal antibody. In another embodiment, the antibody is a monoclonal antibody. In still a further embodiment, the antibody is a teleost antibody. In yet another embodiment, the antibody is a salmon antibody. In still another embodiment, the antibody is an IgM antibody. In yet another embodiment, the antibody is a chimeric antibody.
In another aspect, the invention provides an immunogenic composition comprising a killed virus comprising a PRV polypeptide. In still another aspect, the invention provides an immunogenic composition comprising an attenuated virus comprising a PRV polypeptide. In one embodiment, any of the immunogenic compositions described herein further comprise at least one excipient, additive or adjuvant. In one embodiment, any of the immunogenic compositions described herein further comprise at least one polypeptide, or fragment thereof, from an additional virus.
In another aspect, the invention provides an immunogenic composition comprising a fusion polypeptide, wherein the fusion polypeptide comprises a PRV polypeptide, a fragment, of a variant thereof and at least one polypeptide, or fragment thereof, from an additional virus. In one embodiment, the additional virus is selected from the group consisting of Sleeping disease virus (SDV); salmon pancreas disease virus (SPDV); infectious salmon anemia (ISAV); Viral hemorrhagic septicemia virus (VHSV); infectious hematopoietic necrosis virus (IHNV); infectious pancreatic necrosis virus (IPNV); spring viremia of carp (SVC); channel catfish virus (CCV); Aeromonas salmonicida; Renibacterium salmoninarum; Moritella viscosis, Yersiniosis; Pasteurellosis; Vibro anguillarum; Vibrio logei; Vibrio ordalii; Vibrio salmonicida; Edwardsiella ictaluri; Edwardsiella tarda; Cytophaga columnari; or Piscirickettsia salmonis. 
In another aspect, the invention provides a method of inducing an immune response in an animal, the method comprising administering any PRV immunogenic composition described herein.
In another aspect, the invention provides a method for preventing, or reducing PRV infection in an animal, the method comprising administering any PRV immunogenic composition described herein.
In another aspect, the invention provides a method for preventing, or reducing PRV infection in an animal, the method comprising administering to the animal any antibody described herein.
In one embodiment, the method of any administration in the methods described herein is oral administration, immersion administration or injection administration.
In yet another aspect, the invention provides for use of any of the immunogenic compositions described herein in the manufacture of a vaccine for the treatment of condition PRV infection in an animal.
In yet another aspect, the invention provides for use of any of the immunogenic compositions described herein in the manufacture of a vaccine for preventing or reducing a condition PRV infection in an animal.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Piscine reovirus (PRV) sequence obtained by pyrosequencing. Assembled sequence data mapped against the concatenated sequences of PRV. Genomic regions identified by BLASTN, BLASTX, FASTX, and FASD are shown in red, blue, green, and orange respectively.
FIG. 2. Phylogenetic analysis of the RNA-dependent RNA-polymerase of Reoviridae. Full length amino acid sequences were aligned using the ClustalX14 implemented on MEGA software (Tamura et al., Mol Biol Evol 24, 1596-1599 (2007)) and refined using T-Coffee (Notredame et al., J Mol Biol 302, 205-217 (2000)) to incorporate protein structure data. Phylogenetic analysis was performed using p-distance as model of amino acid substitution as implemented by ICTV for analysis of the Reoviridae family (Mertens et al., T. Family Reoviridae. 447-454 (Elsevier Academic Press, 2005)). MEGA was used to produce phylogenetic trees, reconstructed through the Neighbor Joining (NJ) method. The statistical significance of a particular tree topology was evaluated by bootstrap re-sampling of the sequences 1000 times.
FIG. 3. Graphical representation of group differences in the log ratio of virus load normalized to a salmon host gene. Nonparametric approaches were used to determine statistical significance for comparisons of the relative viral load among healthy and HSMI-affected farmed fish. Log transformations, which did not normalize log ratio distributions, were nonetheless performed for all samples after calculating L1 (virus)/EF1A (housekeeping) ratios to aid in graphical representation. FIG. 3A shows a comparison of adjusted log ratio in mixed heart and kidney samples from healthy farmed fish and farmed fish with HSMI; *, p<0.0001 (Mann-Whitney U). FIG. 3B shows a comparison of adjusted log ratios in farmed fish without HSMI (healthy farmed fish), in the early phase of an HSMI outbreak, in the middle of an HSMI outbreak, and during the peak of an HSMI outbreak; **, p<0.0005; *, p<0.01 (individual Mann-Whitney U). Adjusted log ratios also differed significantly across all four farmed fish groups (p<0.0001; Kruskal-Wallis).
FIG. 4. In situ hybridization was performed using locked nucleic acid (LNA) probes targeting the L2 segment of the Piscine reovirus. Sections were permeabilized using proteinase K followed by hybridization with digoxigenin (DIG)-labeled LNA probes. Sections were incubated with a mouse monoclonal anti-DIG-horse radish peroxidase and stained using a Tyramide Signal Amplification System. Sections were counterstained with Meyer's hematoxylin solution. FIG. 4A shows heart from HSMI-infected fish (10×). FIG. 4B shows heart from HSMI-infected fish (40×). FIG. 4C shows heart from non-infected fish (40×). FIG. 4D shows heart from a fish infected with salmon pancreas disease virus.
FIG. 5. Phylogenetic analysis of the Lambda-1 ORF of the Aquareovirus and Orthoreovirus. Bayesian phylogenetic analyses of sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σNS (σ1 and σ3 of aquareovirus and orthoreovirus had different genomic organizations) were conducted using BEAST, BEAUti and Tracer analysis software packages. Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states. Colored boxes indicate representatives of different reovirus genera or species. Green, Aquareovirus genus; blue, species I (mammalian orthoreovirus); red, species II (avian orthoreovirus); purple, species III (Nelson Bay orthoreovirus); orange, species IV (reptilian orthoreovirus) and light blue, species V (Baboon orthoreovirus).
FIG. 6. Phylogenetic analysis of the Lambda-2 ORF of the Aquareovirus and Orthoreovirus. Bayesian phylogenetic analyses of sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2, and σNS (σ1 and σ3 of aquareovirus and orthoreovirus had different genomic organizations) were conducted using BEAST, BEAUti and Tracer analysis software packages. Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states. Colored boxes indicate representatives of different reovirus genera or species. Green, Aquareovirus genus; blue, species I (mammalian orthoreovirus); red, species II (avian orthoreovirus); purple, species III (Nelson Bay orthoreovirus); orange, species IV (reptilian orthoreovirus) and light blue, species V (Baboon orthoreovirus).
FIG. 7. Phylogenetic analysis of the Lambda-3 ORF of the Aquareovirus and Orthoreovirus. Bayesian phylogenetic analyses of sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σNS (σ1 and σ3 of aquareovirus and orthoreovirus had different genomic organizations) were conducted using BEAST, BEAUti and Tracer analysis software packages. Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states. Colored boxes indicate representatives of different reovirus genera or species. Green, Aquareovirus genus; blue, species I (mammalian orthoreovirus); red, species II (avian orthoreovirus); purple, species III (Nelson Bay orthoreovirus); orange, species IV (reptilian orthoreovirus) and light blue, species V (Baboon orthoreovirus).
FIG. 8. Phylogenetic analysis of the Mu-1 ORF of the Aquareovirus and Orthoreovirus. Bayesian phylogenetic analyses of sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σNS (σ1 and σ3 of aquareovirus and orthoreovirus had different genomic organizations) were conducted using BEAST, BEAUti and Tracer analysis software packages. Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states. Colored boxes indicate representatives of different reovirus genera or species. Green, Aquareovirus genus; blue, species I (mammalian orthoreovirus); red, species II (avian orthoreovirus); purple, species III (Nelson Bay orthoreovirus); orange, species IV (reptilian orthoreovirus) and light blue, species V (Baboon orthoreovirus).
FIG. 9. Phylogenetic analysis of the Mu-2 ORF of the Aquareovirus and Orthoreovirus. Bayesian phylogenetic analyses of sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σNS (σ1 and σ3 of aquareovirus and orthoreovirus had different genomic organizations) were conducted using BEAST, BEAUti and Tracer analysis software packages. Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states. Colored boxes indicate representatives of different reovirus genera or species. Green, Aquareovirus genus; blue, species I (mammalian orthoreovirus); red, species II (avian orthoreovirus); purple, species III (Nelson Bay orthoreovirus); orange, species IV (reptilian orthoreovirus) and light blue, species V (Baboon orthoreovirus).
FIG. 10. Phylogenetic analysis of the Mu-3 ORF of the Aquareovirus and Orthoreovirus. Bayesian phylogenetic analyses of sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σNS (σ1 and σ3 of aquareovirus and orthoreovirus had different genomic organizations) were conducted using BEAST, BEAUti and Tracer analysis software packages. Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states. Colored boxes indicate representatives of different reovirus genera or species. Green, Aquareovirus genus; blue, species I (mammalian orthoreovirus); red, species II (avian orthoreovirus); purple, species III (Nelson Bay orthoreovirus); orange, species IV (reptilian orthoreovirus) and light blue, species V (Baboon orthoreovirus).
FIG. 11. Phylogenetic analysis of the Sigma-2 ORF of the Aquareovirus and Orthoreovirus. Bayesian phylogenetic analyses of sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σNS (σ1 and σ3 of aquareovirus and orthoreovirus had different genomic organizations) were conducted using BEAST, BEAUti and Tracer analysis software packages. Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states. Colored boxes indicate representatives of different reovirus genera or species. Green, Aquareovirus genus; blue, species I (mammalian orthoreovirus); red, species II (avian orthoreovirus); purple, species III (Nelson Bay orthoreovirus); orange, species IV (reptilian orthoreovirus) and light blue, species V (Baboon orthoreovirus).
FIG. 12. Phylogenetic analysis of the Sigma-NS ORF of the Aquareovirus and Orthoreovirus. Bayesian phylogenetic analyses of sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σNS (σ1 and σ3 of aquareovirus and orthoreovirus had different genomic organizations) were conducted using BEAST, BEAUti and Tracer analysis software packages. Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states. Colored boxes indicate representatives of different reovirus genera or species. Green, Aquareovirus genus; blue, species I (mammalian orthoreovirus); red, species II (avian orthoreovirus); purple, species III (Nelson Bay orthoreovirus); orange, species IV (reptilian orthoreovirus) and light blue, species V (Baboon orthoreovirus).
FIG. 13. Putative ORF of 51 has characteristics similar to FAST proteins. Hydrophobicity plots of ARV (red) and PRV (blue) obtained using the Kyle-Doolittle algorithm implemented in the program TopPred (available at http://mobyle.pasteur fr/cgibin/portal.py?form=toppred). Sequence analysis show that PRV contains the primary components of a FAST protein: hydrophobic region (HP), transmembrane domain (TM) and basic region (BR).
FIG. 14. The pathology of PRV infection can include liver discoloration, heamopericardium, congestion in fatty tissue and swollen spleen.
FIG. 15. Coverage by pyrosequencing.
FIG. 16. Phylogenetic analysis of PRV, Orthoreovirus and Aquareovirus.
FIG. 17. Diagnosis of HSMI showing infiltration of the epicardium as well as severe inflammation of the myocardium.
FIG. 18. A schematic illustration for a method for generating antibodies against σ1, σ3 and μ1C. FIG. 18A shows outer capsid proteins σ1, σ3, λ2, μ1c and inner capsid proteins λ1, σ2, ρ2, and λ3. FIG. 18B shows amplification of σ1, σ3 and μ1C full length segments by PCR. FIG. 18C shows that the amplified segments can be cloned into an expression vector to make an expression construct. The expression can be used to express antigens in an expression system (e.g. E. coli). The antigens can then be purified and used to immunize rabbits.
FIG. 19. Peptide antigen. FIG. 19A shows FAST (fusion-associated small transmembrane protein encoded by S4. FIG. 19B shows the variation of the antigenic index as a function of amino acid position. The higher the antigenic index, the more likely should be that antibodies would “see” those groups of residues.
FIG. 20. The antiserum recognizes the μ1C protein as found in Western blots of E. coli His-tag fusion protein. Lines 11-13, eluates of purified protein; L14-15, dilutions of pellet of induced bacteria, L16-L17 pellet of non-induced bacteria
FIG. 21. The antiserum recognizes the σ2 protein as found in western blots of E. coli His-tag fusion protein and different negative controls. Lines 2-4, eluates of purified protein; L5-6, dilutions of pellet of induced bacteria, L7-L8 pellet of non-induced bacteria.
FIG. 22. PRV Illustration.



DETAILED DESCRIPTION
The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%.
As used herein, “PRV” refers to isolates of the Piscine reoviruses described herein.
As used herein, the term “animal” refers to a vertebrate, including, but not limited to a teleost (e.g. salmon).
As used herein, the term “PRV” polypeptide includes a PRV polypeptide, a PRV polypeptide fragment or a PRV polypeptide variant, or a polypeptide substantially identical to a PRV polypeptide.
As used herein, the term “antibody” refers to an antibody that binds to a PRV polypeptide, a PRV polypeptide fragment or a PRV polypeptide variant, or a polypeptide substantially identical to a PRV polypeptide and inhibit, neutralize or reduce the activity or function of a PRV polypeptide or a PRV. The term antibody specifically excludes diagnostic antibodies which bind a PRV polypeptide, a PRV polypeptide fragment or a PRV polypeptide variant, or a polypeptide substantially identical to a PRV polypeptide and which do not inhibit, neutralize or reduce the activity or function of the polypeptide or the PRV.
Mariculture, aquaculture in marine environments, is an increasingly important source of dietary protein for human consumption. HSMI appears 5 to 9 months after fish are transferred from fresh water to ocean pens (Kongtorp et al., J Fish Dis 27, 351-358 (2004)), but outbreaks have been recorded as early as 14 days following seawater transfer. Affected fish are anorexic and display abnormal swimming behavior. Autopsy findings typically include a pale heart, yellow liver, ascites, swollen spleen and petechiae in the perivisceral fat. The pathology is further characterized by epi-, endo- and myocarditis, myocardial necrosis, myositis and necrosis of red skeletal muscle, and up to 20% mortality (Kongtorp et al., Dis Aquat Organ 59, 217-224 (2004)). While mortality is variable (up to 20%), morbidity may be very high in affected cages. HSMI is diagnosed on the basis of histopathology. The major pathological changes occur in the myocardium and red skeletal muscle, where extensive inflammation and multifocal necrosis of myocytes are evident.
Disease can be induced in naïve fish by experimental injection with tissue homogenate from HSMI diseased fish or by cohabitation with fish with HSMI (Kongtorp et al., J Fish Dis 27, 351-358 (2004)). Virus-like particles have been observed (Watanabe, K. et al., Dis Aquat Organ 70, 183-192 (2006)); however, efforts to implicate an infectious agent by using culture, subtractive cloning and consensus polymerase chain reaction have been unsuccessful.
In one aspect, the present invention shows that HSMI is associated with infection with a novel reovirus termed Piscine reovirus (PRV). PRV was identified through high-throughput pyrosequencing of serum and heart tissue of experimentally infected fish using novel frequency analysis methods as well as standard alignment methods. In another aspect, the present invention provides PRV nucleic acid sequences.
In other aspects, the invention is directed to expression constructs, for example plasmids and vectors, and isolated nucleic acids which comprise PRV nucleic acid sequences of SEQ ID NOs: 1-10, fragments, complementary sequences, and/or variants thereof.
The nucleic acid sequences and polypeptides described herein may be useful for multiple applications, including, but not limited to, generation of antibodies and generation of immunogenic compositions. For example, in one aspect, the invention is directed to an immunogenic composition comprising a polypeptide encoded by a PRV nucleic sequence acid of any one of SEQ ID NOs: 1-10.
In another aspect, the invention is directed to an immunogenic composition comprising a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 29-40.
In one aspect, the invention provides an isolated PRV nucleic acid having the sequence of any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV nucleic acid which comprises consecutive nucleotides having a sequence selected from the group consisting of any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV nucleic acid which comprises consecutive nucleotides having a sequence selected from a variant of any of SEQ ID NOs: 1-10 or a fragment thereof. In one embodiment, the variant has at least about 85% identity to SEQ ID NOs: 1-10, or a fragment thereof. In one embodiment of the above aspect of the invention, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any one of SEQ ID NOs: 1-10, or a fragment thereof.
In one aspect, the invention provides an isolated PRV nucleic acid complementary to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV nucleic acid which comprises consecutive nucleotides complementary to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV nucleic acid which comprises consecutive nucleotides complementary to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof. In one embodiment, the variant has at least about 85% identity to SEQ ID NOs: 1-10, or a fragment thereof. In one embodiment of the above aspect of the invention, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any one of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV nucleic acid having a sequence substantially identical to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV nucleic acid having a sequence substantially identical to a sequence complementary to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
The PRV nucleic acid sequences described herein may be useful for, inter alia, expression of PRV-encoded proteins or fragments, variants, or derivatives thereof, generation of antibodies against PRV proteins, generating vaccines against Piscine reoviruses, and screening for drugs effective against Piscine reoviruses as described herein.
In one aspect, the invention provides an isolated PRV polypeptide encoded by a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In one embodiment, the PRV polypeptide fragment can be a polypeptide comprising about 8 consecutive amino acids of a PRV polypeptide described herein. In another embodiment, the fragment can be a polypeptide comprising about 10 consecutive amino acids of a PRV polypeptide described herein. In another embodiment, the fragment can be a polypeptide comprising about 14 consecutive amino acids of a PRV polypeptide described herein. In another embodiment, the fragment can be a polypeptide comprising about 16 consecutive amino acids of a PRV polypeptide described herein. In another embodiment, the fragment can be a polypeptide comprising about 18 consecutive amino acids of a PRV polypeptide described herein. In another embodiment, the fragment can be a polypeptide comprising about 20 consecutive amino acids of a PRV polypeptide described herein. In another embodiment, the fragment can be a polypeptide comprising about 21 or more consecutive amino acids of a PRV polypeptide described herein.
In yet another embodiment, the PRV polypeptide fragment can be a polypeptide comprising from about 8 to about 50, about 8 to about 100, about 8 to about 200, about 8 to about 300, about 8 to about 400, about 8 to about 500, about 8 to about 600, about 8 to about 700, about 8 to about 800, about 8 to about 900 or more consecutive amino acids from a PRV polypeptide.
In another aspect, the invention provides an isolated PRV polypeptide encoded by a nucleic acid which comprises consecutive nucleotides having a sequence selected from a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV polypeptide encoded by a nucleic acid which comprises consecutive nucleotides having a sequence selected from a variant of a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10 or a fragment thereof. In one embodiment, the variant has at least about 85% identity to SEQ ID NOs: 1-10, or a fragment thereof. In one embodiment of the above aspect of the invention, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any one of SEQ ID NOs: 1-10, or a fragment thereof.
In one aspect, the invention provides an isolated PRV polypeptide encoded by a nucleic acid complementary a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV polypeptide encoded by a nucleic acid which comprises consecutive nucleotides a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV polypeptide encoded by a nucleic acid having a sequence substantially identical to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV polypeptide encoded by a nucleic acid having a sequence substantially identical to a sequence complementary to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In one aspect, the invention provides an isolated PRV polypeptide having the sequence of any of SEQ ID NOs: 29-40, or a fragment thereof.
In another aspect, the invention provides an isolated PRV polypeptide which comprises consecutive amino acids having a sequence selected from the group consisting of any of SEQ ID NOs: 29-40, or a fragment thereof.
In another aspect, the invention provides an isolated PRV polypeptide which comprises consecutive amino acids having a sequence selected from a variant of any of SEQ ID NOs: 29-40, or a fragment thereof. In one embodiment, the variant has at least about 85% identity to SEQ ID NOs: 29-40, or a fragment thereof. In one embodiment of the above aspect of the invention, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any one of SEQ ID NOs: 1-10, or a fragment thereof.
In another aspect, the invention provides an isolated PRV polypeptide having a sequence substantially identical to a PRV amino acid sequence in any of SEQ ID NOs: 29-40, or a fragment thereof.
The PRV polypepetides and amino acid sequences described herein may be useful for, inter alia, expression of PRV-encoded proteins or fragments, variants, or derivatives thereof, and generating vaccines against Piscine reoviruses.
In one aspect, the invention provides an isolated PRV polypeptide encoded by a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, or a fragment thereof.
In one embodiment, the isolated PRV polypeptide fragment can be a polypeptide comprising about 8 consecutive amino acids of a PRV amino acid sequence of any of SEQ ID NOs: 29-40. In another embodiment, the fragment can be a polypeptide comprising about 10 consecutive amino acids of a PRV amino acid sequence of any of SEQ ID NOs: 29-40. In another embodiment, the fragment can be a polypeptide comprising about 14 consecutive amino acids of a PRV amino acid sequence of any of SEQ ID NOs: 29-40. In another embodiment, the fragment can be a polypeptide comprising about 16 consecutive amino acids of a PRV amino acid sequence of any of SEQ ID NOs: 29-40. In another embodiment, the fragment can be a polypeptide comprising about 18 consecutive amino acids of a PRV amino acid sequence of any of SEQ ID NOs: 29-40. In another embodiment, the fragment can be a polypeptide comprising about 20 consecutive amino acids of a PRV amino acid sequence of any of SEQ ID NOs: 29-40. In another embodiment, the fragment can be a polypeptide comprising about 21 or more consecutive amino acids of a PRV amino acid sequence of any of SEQ ID NOs: 29-40.
In yet another embodiment, the isolated PRV polypeptide fragment can be a polypeptide comprising from about 8 to about 50, about 8 to about 100, about 8 to about 200, about 8 to about 300, about 8 to about 400, about 8 to about 500, about 8 to about 600, about 8 to about 700, about 8 to about 800, about 8 to about 900 or more consecutive amino acids of a PRV amino acid sequence of any of SEQ ID NOs: 29-40.
In another aspect, the invention provides an isolated PRV polypeptide which comprises consecutive amino acids having a sequence selected from a PRV amino acid sequence of any of SEQ ID NOs: 29-40.
In another aspect, the invention provides an isolated PRV polypeptide which comprises consecutive nucleotides having a sequence selected from a variant a PRV amino acid sequence of any of SEQ ID NOs: 29-40, or a fragment thereof. In one embodiment, the variant has at least about 85% identity to any of SEQ ID NOs: 29-40, or a fragment thereof. In one embodiment of the above aspect of the invention, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to any of SEQ ID NOs: 29-40, or a fragment thereof.
In another aspect, the invention provides an isolated PRV polypeptide substantially identical to variant a PRV amino acid sequence of any of SEQ ID NOs: 29-40, or a fragment thereof.
“Substantially identical,” in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least of at least 98%, at least 99% or higher nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Thus, in certain embodiments, polypeptides that a substantially identical to the PRV polypeptides described herein can also be used to generate antibodies that bind to the PRV polypeptides described herein.
“Percent identity” in the context of two or more nucleic acids or polypeptide sequences, refers to the percentage of nucleotides or amino acids that two or more sequences or subsequences contain which are the same. A specified percentage of amino acid residues or nucleotides can have a specified identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. In one aspect, the invention provides a PRV polypeptide which is a variant of a PRV polypeptide and has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to a PRV polypeptide shown in SEQ ID NOs 29-40.
It will be understood that, for the particular PRV polypeptides described here, natural variations can exist between individual PRV strains. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al in “The Proteins” Academic Press New York (1979). Amino acid replacements between related amino 15 acids or replacements which have occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington, D.C., 1978, vol. 5, suppl. 3). Other amino acid substitutions 20 include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val and Ala/Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science, 227, 1435-1441, 1985) and determining the functional similarity between homologous proteins. Such amino acid substitutions of the exemplary embodiments of this invention, as well as variations having deletions and/or insertions are within the scope of the invention as long as the resulting proteins retain their immune reactivity. It is know that polypeptide sequences having one or more amino acid sequence variations as compared to a reference polypeptide may still be useful for generating antibodies that bind the reference polypeptide. Thus in certain embodiments, the PRV polypeptides and the antibodies and antibody generation methods related thereto encompass PRV polypeptides isolated from different virus isolates that have sequence identity levels of at least about 90%, while still representing the same PRV protein with the same immunological characteristics.
The sequence identities can be determined by analysis with a sequence comparison algorithm or by a visual inspection. Protein and/or nucleic acid sequence identities (homologies) can be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST and BLAST 2.2.2. or FASTA version 3.0t78 algorithms and the default parameters discussed below can be used.
An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the FASTA algorithm, which is described in Pearson, W. R. & Lipman, D. J., Proc. Natl. Acad. Sci. U.S.A. 85: 2444, 1988. See also W. R. Pearson, Methods Enzymol. 266: 227-258, 1996. Exemplary parameters used in a FASTA alignment of DNA sequences to calculate percent identity are optimized, BL50 Matrix 15: −5, k-tuple=2; joining penalty=40, optimization=28; gap penalty −12, gap length penalty=−2; and width=16.
Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www ncbi.nlm.nih.gov/). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, less than about 0.01, and less than about 0.001.
Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984.
Another example of an algorithm that is suitable for multiple DNA and amino acid sequence alignments is the CLUSTALW program (Thompson, J. D. et al., Nucl. Acids. Res. 22:4673-4680, 1994). ClustalW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties were 10 and 0.05 respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919, 1992).
In yet a further aspect, the invention provides a computer readable medium having stored thereon (i) a nucleic acid sequence selected from the group consisting of: a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, a sequence substantially identical to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10; a sequence variant of a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10; or (ii) an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of: a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10, an amino acid sequence encoded by a sequence substantially identical to a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10; an amino acid sequence encoded by a sequence variant of a PRV nucleic acid sequence in any of SEQ ID NOs: 1-10.
The polypeptides described herein can be used for raising antibodies (e.g. for vaccination purposes). In one aspect, the invention provides antibody that binds a PRV polypeptide, a PRV polypeptide fragment or a PRV polypeptide variant, or a polypeptide substantially identical to a PRV polypeptide and wherein the antibody is a vaccine antibody that inhibits, neutralizes or reduces the activity or function of the polypeptide or a PRV. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a teleost antibody or a chimeric antibody. Methods for purifying immunoglobulins from teleosts are also known in the art. See, for example, Havarstein et al, Dev Comp Immunol 1988, 12(4):773-85; Al-Harbi et al, Bull Eur Ass Fish Pathol 1993, 13:40-4; Itami et al, Nippon Suisan Gakkaishi 1988, 54(9):1611-7.
In still a further aspect, the invention provides a PRV immunogenic composition comprising a PRV polypeptide, a PRV polypeptide fragment or a PRV polypeptide variant, or a polypeptide substantially identical to a PRV polypeptide.
As used herein, the term “immunogenic polypeptide” refers to a PRV polypeptide, or a fragment of a PRV polypeptide capable of inducing an immune response in a vertebrate host (e.g. a teleost). The term “immunogenic polypeptide” also refers to a PRV polypeptide, or a fragment of a PRV polypeptide that can be used to generate an antibody against the PRV polypeptide, or a fragment of a PRV polypeptide using other antibody generation techniques known in the art, including, but not limited to, hybridoma, phage display and ribosome display technologies.
In still a further aspect, the invention provides a PRV vaccine composition comprising a PRV nucleic acid, a PRV nucleic acid fragment or a PRV nucleic acid variant, a nucleic acid substantially identical to a PRV nucleic acid, a PRV polypeptide, a PRV polypeptide fragment or a PRV polypeptide variant, or a polypeptide substantially identical to a PRV polypeptide.
One of skill in the art will recognize that when polypeptides are used for raising antibodies, it is not necessary to use the entire polypeptide to generate an antibody capable of recognizing the full length polypeptide. In certain aspects, the invention is directed to methods for generating antibodies that bind to the PRV polypeptides described herein by generating antibodies that bind to a fragment of a polypeptide described herein. Thus, in one aspect, the invention relates to vaccines for combating PRV infection, that comprise a protein or immunogenic fragments of a PRV polypeptide. Still another embodiment of the present invention relates to the PRV proteins described herein or immunogenic fragments thereof for use in a vaccine. In still another embodiment, the invention relates to the use of the PRV proteins described herein or immunogenic fragments thereof for the manufacturing of a vaccine for combating PRV infections.
In one embodiment, the PRV immunogenic compositions and PRV vaccines described herein are capable of ameliorating the symptoms of a PRV infection and/or of reducing the duration of a PRV infection. In another embodiment, the immunogenic compositions are capable of inducing protective immunity against PRV infection. The immunogenic compositions of the invention can be effective against the PRV disclosed herein, and may also be cross-reactive with, and effective against, multiple different clades and strains of PRV, and against other reoviruses.
In other aspect, the invention provides a nucleic acid vectors comprising a PRV nucleic acid sequence, a PRV nucleic acid fragment or a PRV nucleic acid variant, or a nucleic acid substantially identical to a PRV nucleic acid.
In another aspect, the invention provides a nucleic acid vector encoding a PRV polypeptide, a PRV polypeptide fragment or a PRV polypeptide variant, or a polypeptide substantially identical to a PRV polypeptide. Non-limiting examples of vectors include, but are not limited to retroviral, adenoviral, adeno-associated viral, lentiviral, and vesiculostomatitis viral vectors.
In yet another aspect, the invention provides a host organism comprising a nucleic acid vector encoding a PRV polypeptide, a PRV polypeptide fragment or a PRV polypeptide variant, or a polypeptide substantially identical to a PRV polypeptide. In one embodiment, the host organism is a prokaryote, a eukaryote, or a fungus. In another embodiment the organism is a teleost (e.g. a salmon).
In another aspect, the invention provides a method of inducing an immune response in an animal (e.g. a salmon), the method comprising administering a PRV nucleic acid, a PRV polypeptide or a PRV immunogenic composition to the animal. Methods for administering polypeptides to animals (e.g. teleosts), and methods for generating immune responses in animals (e.g. teleosts) by administering immunogenic peptides in immunogenically effective amounts are known in the art.
Teleost lack bone marrow or lymph and B-cell lymphogenesis occurs in the head kidney (pronephros) and spleen. For a review of preimmune diversification and antibody generation in teleosts, see Solem and Stenvik, Developmental and Comparative Immunology 30 (2006) 57-76). Unlike mammals, where several classes of immunoglobulins (e.g. IgG, IgE and IgA, among others) are present in the circulation, structurally heterogeneous IgM tetramers are the most prevalent immunoglobulin in teleosts (Warr G. W. (1995): Developmental and Comparative Immunology, 19, 1-12; Koumansvandiepen et al, (1995) Developmental and Comparative Immunology, 19, 97-108; Kaattari et al, 1998. Immunol. Rev. 166:133-142; Evans et al, 1998. J. Theor. Biol. 195:505-524). IgD, IgZ, IgT and IgH immunoglobulins have also been identified in teleosts (Hordvik et al, (1999) Scandinavian Journal of Immunology, 50, 202-2101; Hirono et al, (2003) Fish & Shellfish Immunology, 15, 63-70; Danilova et al, (2000) Immunogenetics, 52, 81-91; Hansen et al, (1994) Molecular Immunology, 31, 499-501; Svyan et al, (2005) European Journal of Immunology, 35, 3320-3331).
The polypeptides described herein can be used in the form of a PRV immunogenic composition to vaccinate an animal (e.g. a teleost) according to any method known in the art. See, for example, Veseley et al, Veterinarni Medicina, 51, 2006 (5): 296-302; Engelbrecht et al, Acta Vet Scand 1997, 38(3):275-82; Ingram et al, J Fish Biol 1979, 14(3):249-60. An immunogenic composition for use in vaccination can also include attenuated live viral vaccines, inactivated (killed) viral vaccines, and subunit vaccines. In certain embodiments, PRVs may be attenuated by removal or disruption of viral sequences whose products cause or contribute to the disease and symptoms associated with PRV infection, and leaving intact those sequences required for viral replication. In this way an attenuated PRV can be produced that replicates in animals, and induces an immune response in animals, but which does not induce the deleterious disease and symptoms usually associated with PRV infection. One of skill in the art can determine which PRV sequences can or should be removed or disrupted, and which sequences should be left intact, in order to generate an attenuated PRV suitable for use as a vaccine. PRV vaccines may also comprise inactivated PRV, such as by chemical treatment, to “kill” the viruses such that they are no longer capable of replicating or causing disease in animals, but still induce an immune response in an animal (e.g. a salmon). There are many suitable viral inactivation methods known in the art and one of skill in the art can readily select a suitable method and produce an inactivated “killed” PRV suitable for use as a vaccine.
Methods of purification of polypeptides and of inactivated virus are known in the art and may include one or more of, for instance gradient centrifugation, ultracentrifugation, continuous-flow ultracentrifugation and chromatography, such as ion exchange chromatography, size exclusion chromatography, and liquid affinity chromatography. Additional method of purification include ultrafiltration and dialfiltration. See J P Gregersen “Herstellung von Virussimpfstoffen aus Zellkulturen” Chapter 4.2 in Pharmazeutische Biotechnology (eds. O. Kayser and R H Mueller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000. See also, O'Neil et al., “Virus Harvesting and Affinity Based Liquid Chromatography. A Method for Virus Concentration and Purification”, Biotechnology (1993) 11:173-177; Prior et al., “Process Development for Manufacture of Inactivated HIV-1”, Pharmaceutical Technology (1995) 30-52; and Majhdi et al., “Isolation and Characterization of a Coronavirus from Elk Calves with diarrhea” Journal of Clinical Microbiology (1995) 35(11): 2937-2942.
Other examples of purification methods suitable for use in the invention include polyethylene glycol or ammonium sulfate precipitation (see Trepanier et al., “Concentration of human respiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration” Journal of Virological Methods (1981) 3(4):201-211; Hagen et al., “Optimization of Poly(ethylene glycol) Precipitation of Hepatitis Virus Used to prepare VAQTA, a Highly Purified Inactivated Vaccine” Biotechnology Progress (1996) 12:406-412; and Carlsson et al., “Purification of Infectious Pancreatic Necrosis Virus by Anion Exchange Chromatography Increases the Specific Infectivity” Journal of Virological Methods (1994) 47:27-36) as well as ultrafiltration and microfiltration (see Pay et al., Developments in Biological Standardization (1985) 60:171-174; Tsurumi et al., “Structure and filtration performances of improved cuprammonium regenerated cellulose hollow fiber (improved BMM hollow fiber) for virus removal” Polymer Journal (1990) 22(12):1085-1100; and Makino et al., “Concentration of live retrovirus with a regenerated cellulose hollow fiber, BMM”, Archives of Virology (1994) 139(1-2):87-96.).
Polypeptides and viruses can be purified using chromatography, such as ion exchange, chromatography. Chromatic purification allows for the production of large volumes of virus containing suspension. The viral product of interest can interact with the chromatic medium by a simple adsorption/desorption mechanism, and large volumes of sample can be processed in a single load. Contaminants which do not have affinity for the adsorbent pass through the column. The virus material can then be eluted in concentrated form.
Anion exchange resins that may be used include DEAE, EMD TMAE. Cation exchange resins may comprise a sulfonic acid-modified surface. Viruses can be purified using ion exchange chromatography comprising a strong anion exchange resin (e.g. EMD TMAE) for the first step and EMD-SO3 (cation exchange resin) for the second step. A metal-binding affinity chromatography step can optionally be included for further purification. (See, e.g., WO 97/06243).
A resin such as Fractogel EMD can also be used This synthetic methacrylate based resin has long, linear polymer chains covalently attached and allows for a large amount of sterically accessible ligands for the binding of biomolecules without any steric hindrance.
Column-based liquid affinity chromatography is another purification method that can be used invention. One example of a resin for use in purification method is Matrex Cellufine Sulfate (MCS). MCS consists of a rigid spherical (approx. 45-105 μm diameter) cellulose matrix of 3,000 Dalton exclusion limit (its pore structure excludes macromolecules), with a low concentration of sulfate ester functionality on the 6-position of cellulose. As the functional ligand (sulfate ester) is relatively highly dispersed, it presents insufficient cationic charge density to allow for most soluble proteins to adsorb onto the bead surface. Therefore the bulk of the protein found in typical virus pools (cell culture supernatants, e.g. pyrogens and most contaminating proteins, as well as nucleic acids and endotoxins) are washed from the column and a degree of purification of the bound virus is achieved.
Inactivated viruses may be further purified by gradient centrifugation, or density gradient centrifugation. For commercial scale operation a continuous flow sucrose gradient centrifugation would be an option. This method can be used to purify antiviral vaccines and is known to one skilled in the art.
Additional purification methods which may be used to purify viruses of the invention include the use of a nucleic acid degrading agent, a nucleic acid degrading enzyme, such as a nuclease having DNase and RNase activity, or an endonuclease, such as from Serratia marcescens, membrane adsorbers with anionic functional groups or additional chromatographic steps with anionic functional groups (e.g. DEAE or TMAE). An ultrafiltration/dialfiltration and final sterile filtration step could also be added to the purification method.
The purified immunogenic preparations described herein can be substantially free of contaminating proteins derived from the cells or cell culture and can comprise less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/μg virus antigen, and less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/dose.
In one aspect, vaccination of animals may be performed by directly injecting the PRV polypeptides, fragments or variants thereof into the animal to generate an immunogenic response. In certain embodiments, the PRV polypeptides can be injected by themselves, or as immunogenic PRV compositions comprising other components, including, for example, excipients, additives and adjuvants.
To produce the immunogenic preparations described herein, the PRV nucleic acid sequences of the invention can be delivered to cultured cells, for example by transfecting cultured cells with plasmids or expression vectors containing PRV nucleic acid sequences, or by infecting cultured cells with recombinant viruses containing PRV nucleic acid sequences. PRV polypeptides may then be expressed in a host cell or expression system and purified. A host cell may be a cell of bacterial origin, e.g. Escherichia coli, Bacillus subtilis and Lactobacillus species, in combination with bacteria-based plasmids as pBR322, or bacterial expression vectors as pGEX, or with bacteriophages. The host cell may also be of eukaryotic origin, e.g. yeast-cells in combination with yeast-specific vector molecules, or higher eukaryotic cells like insect cells (Luckow et al; Bio-technology 6: 47-55 (1988)) in combination with vectors or recombinant baculoviruses, plant cells in combination with e.g. Ti-plasmid based vectors or plant viral vectors (Barton, K. A. et al; Cell 32: 1033 (1983), mammalian cells like Hela cells, Chinese Hamster Ovary cells (CHO) or Crandell Feline Kidney-cells, also with appropriate vectors or recombinant viruses. In vitro expression systems, such as in-vitro transcription and in-vitro translation systems can also be used to generate the PRV polypeptides described herein. The purified proteins can then be incorporated into compositions suitable for administration to animals. Methods and techniques for expression and purification of recombinant proteins are well known in the art, and any such suitable methods may be used.
Vaccination may also be performed by direct vaccination with a DNA encoding a PRV polypeptide. When using such vaccines, the nucleic acid is administered to the animal, and the immunogenic polypeptide(s) encoded by the nucleic acid are expressed in the animal, such that an immune response against the proteins or peptides is generated in the animal. Subunit vaccines may also be proteinaceous vaccines, which contain the viral proteins or subunits themselves, or portions of those proteins or subunits. Any suitable plasmid or expression vector capable of driving expression of a polypeptide may be used. Plasmids and expression vectors can include a promoter for directing transcription of the nucleic acid. The nucleic acid sequence encoding PRV polypeptides may also be incorporated into a suitable recombinant virus for administration to the animal. Examples of suitable viruses include, but are not limited to, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses. One of skill in the art will be able to select a suitable plasmid, expression vector, or recombinant virus for delivery of the PRV nucleic acid sequences of the invention. Direct vaccination with DNA encoding proteins has been successful for many different proteins. (As reviewed in e.g. Donnelly et al. The Immunologist 2: 20-26 (1993)).
Vaccination with the PRV nucleic acids and polypeptides described herein can also be performed using live recombinant carriers capable of expressing the polypeptides described herein. Live recombinant carriers are micro-organisms or viruses in which additional genetic information, e.g. a nucleic acid sequence encoding a PRV polypeptide, or a fragment thereof has been cloned. Fish infected with such live recombinant carriers will produce an immunological response not only against the immunogens of the carrier, but also against the PRV polypeptide or PRV polypeptide fragment. Non-limiting examples of live recombinant carriers suitable for use with the methods described herein includes Vibrio anguillarum (Singer, J. T. et al. New Developments in Marine Biotechnology, p. 303-306, Eds. Le Gal and Halvorson, Plenum Press, New York, 1998), and alphavirus-vectors (Sondra Schlesinger and Thomas W. Dubensky Jr. Alphavirus vectors for gene expression and vaccines. Current opinion in Biotechnology, 10:434439 (1999)
Alternatively, passive vaccination can be performed by raising PRV antibodies in a first animal species (e.g. a rabbit), from antibody-producing cell lines, or from in-vitro techniques before administering such antibodies (in purified or unpurified form) to second animal species (e.g. a teleost). This type of passive vaccination can be used when the second animal is already infected with a PRV. In some cases, passive vaccination can be useful where the infection in the second animal cannot, or has not had sufficient time to mount an immune response to the infection.
Many methods for the vaccination of teleosts are known in the art. For example. Vaccination with the PRV nucleic acids and polypeptides described herein can be performed in teleosts by injection, immersion, dipping or through oral administration. The administration protocol can be optimized in accordance with standard vaccination practice
For oral vaccination of teleosts, the PRV nucleic acids, polypeptides or immunogenic compositions described herein can be mixed with feed, coated on the feed or be administered in an encapsulated form. In certain embodiments, vaccination may be performed by incubating live feed such as Artemia nauplii, copepods or rotifers in a PRV vaccine suspension prior to feeding an animal (e.g. a teleost) such that ingestion of the live feed will cause the PRV vaccine to accumulate in the digestive tract of the animal undergoing vaccination. One skilled in the art will appreciate that these methods of administration may expose an antigen to potential breakdown or denaturation and thus the skill artisan will ensure that the method of vaccination will be appropriate for a chosen antigen. In the case of oral vaccination, the vaccine may also be mixed with one or more carriers. Carriers suitable for use in oral vaccination include both metabolizable and non-metabolizable substances.
Vaccination of teleosts can also be performed by immersion protocols Skin and gill epithelia in fish have mucosal surfaces that contribute to the recognition of pathogens by adsorbing antigens. Adsorption in turn results in the activation of antibody producing cells as part of the immune response. Thus in one embodiment, vaccination of fish with the polypeptides described herein can be performed by immersing fish in water containing a PRV vaccine composition. At least two types of immersion vaccination can be used in conjunction with the polypeptides described herein. In dip vaccination, fish are immersed in water comprising for a short period of time (e.g. about 30 seconds) in a concentrated vaccine solution (e.g. 1 part vaccine, 9 parts water). In bath vaccination, immersion occurs for longer periods of time (e.g. several hours) in water containing lower vaccine concentrations. One skilled in the art will readily be able to determine the dilution of PRV vaccine and the duration of immersion sufficient to induce a immune reaction in an immersion protocol.
Another method for vaccinating teleosts with the PRV nucleic acids and polypeptides described herein is by injection vaccination. In injection vaccination, a vaccine is injected into the abdominal cavity of the fish. Although one skilled in the art can readily determine the proper injection point, a common site for needle insertion in salmon is the midline of the abdomen, one pelvic fin length in front of the base of the pelvic fins. In certain embodiments, the PRV nucleic acids, polypeptides or immunogenic compositions can be delivered into the body cavity of the fish in an oil emulsion, or other adjuvants or additives that enhance and/or prolong immune responses. In addition to intraperitoneal injection, injection vaccination can also be performed by intramuscular injection. One skilled in the art will appreciate that improper handling and needle insertion can cause mortality of fish and thus light anesthesia may be used during the vaccination process to reduce stress and mechanical injury to the animals. The skilled artisan will also appreciate that needles having the proper length and thickness can be important to ensure proper vaccination while avoiding secondary complications due to infection, inflammation or tissue damage.
The PRV nucleic acids, polypeptides or immunogenic compositions described herein can also be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The PRV nucleic acids, polypeptides or immunogenic compositions described herein can be administered in any immunologically effective amount sufficient to trigger an immune response in an animal. In certain instances, this amount can be between about 0.01 and about 1000 micrograms of the PRV nucleic acid, polypeptide or immunogenic composition per animal.
As used herein, the term “immunologically effective amount” refers to an amount capable of inducing, or enhancing the induction of, the desired immune response in an animal. The desired response may include, inter alia, inducing an antibody or cell-mediated immune response, or both. The desired response may also be induction of an immune response sufficient to ameliorate the symptoms of a PRV infection, reduce the duration of a PRV infection, and/or provide protective immunity in an animal against subsequent challenge with a PRV. An immunologically effective amount may be an amount that induces actual “protection” against PRV infection, meaning the prevention of any of the symptoms or conditions resulting from PRV infection in animals. An immunologically effective amount may also be an amount sufficient to delay the onset of symptoms and conditions associated with infection, reduce the degree or rate of infection, reduce in the severity of any disease or symptom resulting from infection, and reduce the viral load of an infected animal.
One of skill in the art can readily determine what is an “immunologically effective amount” of the compositions of the invention without performing any undue experimentation. An effective amount can be determined by conventional means, starting with a low dose of and then increasing the dosage while monitoring the immunological effects. Numerous factors can be taken into consideration when determining an optimal amount to administer, including the size, age, and general condition of the animal, the presence of other drugs in the animal, the virulence of the particular PRV against which the animal is being vaccinated, and the like. The actual dosage is can be chosen after consideration of the results from various animal studies.
The immunologically effective amount of the immunogenic composition may be administered in a single dose, in divided doses, or using a “prime-boost” regimen. The compositions may be administered by any suitable route, including, but not limited to oral, immersion, parenteral, intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, oral, or intraocular routes, or by a combination of routes. The skilled artisan will be able to formulate the vaccine composition according to the route chosen.
In addition to vaccination techniques, antibodies that bind PRV polypeptides described herein can also be generated by any other method known in the art. Exemplary alternative in-vitro antibody generation technologies, transgenic animal technologies and hybridoma technologies. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).
In-vitro technologies suitable for generating PRV binding antibodies include, but are not limited to, ribosome display, yeast display, and bacterial display technologies. Ribosome display is a method of translating mRNAs into their cognate proteins while keeping the protein attached to the RNA. The nucleic acid coding sequence is recovered by RT-PCR (Mattheakis, L. C. et al. 1994. Proc Natl Acad Sci USA 91, 9022). Yeast display is based on the construction of fusion proteins of the membrane-associated alpha-agglutinin yeast adhesion receptor, aga1 and aga2, a part of the mating type system (Broder, et al. 1997. Nature Biotechnology, 15:553-7). Bacterial display is based fusion of the target to exported bacterial proteins that associate with the cell membrane or cell wall (Chen and Georgiou 2002. Biotechnol Bioeng, 79:496-503). In comparison to hybridoma technology, phage and other antibody display methods afford the opportunity to manipulate selection against the antigen target in vitro and without the limitation of the possibility of host effects on the antigen or vice versa.
For example, antibodies that bind PRV polypeptides may be obtained by selecting from libraries, e.g. a phage library. A phage library can be created by inserting a library of random oligonucleotides or a library of polynucleotides containing sequences of interest, such as from the B-cells of an immunized animal (Smith, G. P. 1985. Science 228: 1315-1317). Antibody phage libraries contain heavy (H) and light (L) chain variable region pairs in one phage allowing the expression of single-chain Fv fragments or Fab fragments (Hoogenboom, et al. 2000, Immunol Today 21(8) 371-10). The diversity of a phagemid library can be manipulated to increase and/or alter the immunospecificities of the monoclonal antibodies of the library to produce and subsequently identify additional, desirable, teleost antibodies. For example, the heavy (H) chain and light (L) chain immunoglobulin molecule encoding genes can be randomly mixed (shuffled) to create new HL pairs in an assembled immunoglobulin molecule. Additionally, either or both the H and L chain encoding genes can be mutagenized in a complementarity determining region (CDR) of the variable region of the immunoglobulin polypeptide, and subsequently screened for desirable affinity and neutralization capabilities. Antibody libraries also can be created synthetically by selecting one or more framework sequences and introducing collections of CDR cassettes derived from antibody repertoires or through designed variation (Kretzschmar and von Ruden 2000, Current Opinion in Biotechnology, 13:598-602). The positions of diversity are not limited to CDRs but can also include the framework segments of the variable regions or may include other than antibody variable regions, such as peptides.
Other antibody generation techniques suitable for generating antibodies against the PRV polypeptide, or a fragment of a PRV polypeptide described herein include, the PEPSCAN technique described in Geysen et al (Patent Application WO 84/03564, Patent Application WO 86/06487, U.S. Pat. No. 4,833,092, Proc. Natl. Acad. Sci. 81: 3998-4002 (1984), J. Imm. Meth. 102, 259-274 (1987).
Pepsin or papain digestion of whole antibodies that bind PRV polypeptides can be used to generate antibody fragments that bind PRV polypeptides. In particular, an Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain. An (Fab′)2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. An Fab′ fragment of an antibody molecule can be obtained from (Fab′)2 by reduction with a thiol reducing agent, which yields a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab′ fragments are obtained per antibody molecule treated in this manner.
Antibodies can be produced through chemical crosslinking of the selected molecules (which have been produced by synthetic means or by expression of nucleic acid that encode the polypeptides) or through recombinant DNA technology combined with in vitro, or cellular expression of the polypeptide, and subsequent oligomerization. Antibodies can be similarly produced through recombinant technology and expression, fusion of hybridomas that produce antibodies with different epitope specificities, or expression of multiple nucleic acid encoding antibody variable chains with different epitopic specificities in a single cell.
Antibodies may be either joined directly or indirectly through covalent or non-covalent binding, e.g. via a multimerization domain, to produce multimers. A “multimerization domain” mediates non-covalent protein-protein interactions. Specific examples include coiled-coil (e.g., leucine zipper structures) and alpha-helical protein sequences. Sequences that mediate protein-protein binding via Van der Waals' forces, hydrogen bonding or charge-charge bonds can also be used as multimerization domains. Additional examples include basic-helix-loop-helix domains and other protein sequences that mediate heteromeric or homomeric protein-protein interactions among nucleic acid binding proteins (e.g., DNA binding transcription factors, such as TAFs). One specific example of a multimerization domain is p53 residues 319 to 360 which mediate tetramer formation. Another example is human platelet factor 4, which self-assembles into tetramers. Yet another example is extracellular protein TSP4, a member of the thrombospondin family, which can form pentamers. Additional specific examples are the leucine zippers of jun, fos, and yeast protein GCN4.
Antibodies may be directly linked to each other via a chemical cross linking agent or can be connected via a linker sequence (e.g., a peptide sequence) to form multimers.
The antibodies described herein can be polyclonal or monoclonal. The antibodies can also be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-salmon immunoglobulin). Species specific antibodies avoid certain of the problems associated with antibodies that possess variable and/or constant regions form other species. The presence of such protein sequences form other species can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by an antibody.
The antibodies described herein can be antibodies that bind to other molecules (antigens) via heavy and light chain variable domains, VH and VL, respectively. The antibodies described herein include, but are not limited to IgY, IgY(AFc)), IgG, IgD, IgA, IgM, IgE, and IgL. The antibodies may be intact immunoglobulin molecules, two full length heavy chains linked by disulfide bonds to two full length light chains, as well as subsequences (i.e. fragments) of immunoglobulin molecules, with or without constant region, that bind to an epitope of an antigen, or subsequences thereof (i.e. fragments) of immunoglobulin molecules, with or without constant region, that bind to an epitope of an antigen. Antibodies may comprise full length heavy and light chain variable domains, VH and VL, individually or in any combination.
The basic immunoglobulin (antibody) structural unit can comprise a tetramer. Each tetramer can be composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
The antibodies described herein may exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. In particular, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993) for more antibody fragment terminology). While the Fab′ domain is defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab′ fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. The Fab′ regions may be derived from antibodies of animal or human origin or may be chimeric (Morrison et al., Proc Natl. Acad. Sci. USA 81, 6851-10855 (1984) both incorporated by reference herein) (Jones et al., Nature 321, 522-525 (1986), and published UK patent application No. 8707252, both incorporated by reference herein).
The antibodies described herein can include or be derived from any mammal, such as but not limited to, a fish, a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof and includes isolated fish, human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted or CDR-adapted antibodies, immunoglobulins, cleavage products and other portions and variants thereof. In one embodiment the antibody is purified.
The antibodies described herein include full length antibodies, subsequences (e.g., single chain forms), dimers, trimers, tetramers, pentamers, hexamers or any other higher order oligomer that retains at least a part of antigen binding activity of monomer. Multimers can comprise heteromeric or homomeric combinations of full length antibody, subsequences, unmodified or modified as set forth herein and known in the art. Antibody multimers are useful for increasing antigen avidity in comparison to monomer due to the multimer having multiple antigen binding sites. Antibody multimers are also useful for producing oligomeric (e.g., dimer, trimer, tetramer, etc.) combinations of different antibodies thereby producing compositions of antibodies that are multifunctional (e.g., bifunctional, trifunctional, tetrafunctional, etc.).
Specific examples of antibody subsequences include, for example, Fab, Fab′, (Fab′)2, Fv, or single chain antibody (SCA) fragment (e.g., scFv). Subsequences include portions which retain at least part of the function or activity of full length sequence. For example, an antibody subsequence will retain the ability to selectively bind to an antigen even though the binding affinity of the subsequence may be greater or less than the binding affinity of the full length antibody.
An Fv fragment is a fragment containing the variable region of a light chain VL and the variable region of a heavy chain VH expressed as two chains. The association may be non-covalent or may be covalent, such as a chemical cross-linking agent or an intermolecular disulfide bond (Inbar et al., (1972) Proc. Natl. Acad Sci. USA 69:2659; Sandhu (1992) Crit. Rev. Biotech. 12:437).
Other methods of producing antibody subsequences, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, provided that the subsequences bind to the antigen to which the intact antibody binds.
A single chain antibody (“SCA”) is a genetically engineered or enzymatically digested antibody containing the variable region of a light chain VL and the variable region of a heavy chain, optionally linked by a flexible linker, such as a polypeptide sequence, in either VL-linker-VH orientation or in VH-linker-VL orientation. Alternatively, a single chain Fv fragment can be produced by linking two variable domains via a disulfide linkage between two cysteine residues. Methods for producing scFv antibodies are described, for example, by Whitlow et al., (1991) In: Methods: A Companion to Methods in Enzymology 2:97; U.S. Pat. No. 4,946,778; and Pack et al., (1993) Bio/Technology 11:1271.
The PRV nucleic acids, polypeptides and immunogenic compositions described herein can be used to generate antibodies that that inhibit, neutralize or reduce the activity or function of a polypeptide or a PRV. In certain aspects, the invention is directed to a method for treating an animal (e.g. a salmon), the method comprising administering to the animal PRV nucleic acids, polypeptides and immunogenic compositions, or administering to the animal an antibody which specifically binds to a PRV polypeptide such that the activity or function of a PRV polypeptide or a PRV is inhibited, neutralized or reduced.
In another aspect, the invention described herein relates to PRV immunogenic compositions comprising PRV polypeptides or PRV nucleic acids. In some embodiments, the PRV immunogenic compositions can further comprise carriers, adjuvants, excipients and the like. The PRV immunogenic compositions described herein can be formulated readily by combining the active compounds with immunogenically acceptable carriers well known in the art. The PRV immunogenic compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used to induce an immunogenic response. Such carriers can be used to formulate suitable tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. In one embodiment, the immunogenic composition can be obtained by solid excipient, grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
The immunogenic composition described herein can be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
When a immunogenetically effective amount of a PRV immunogenic composition is administered to an animal, the composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. For example, PRV immunogenic compositions described herein can contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The immunogenic composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The PRV immunogenic compositions can be formulated in aqueous solutions, physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
When the PRV immunogenic compositions is administered orally, protein or other active ingredient of the present invention can be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the immunogenic composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
The PRV immunogenic compositions described herein can encode or contain any of the PRV proteins or peptides described herein, or any portions, fragments, derivatives or mutants thereof, that are immunogenic in an animal. One of skill in the art can readily test the immunogenicity of the PRV proteins and peptides described herein, and can select suitable proteins or peptides to use in subunit vaccines.
The PRV immunogenic compositions described herein comprise at least one PRV amino acid or polypeptide, such as those described herein. The compositions may also comprise one or more additives including, but not limited to, one or more pharmaceutically acceptable carriers, buffers, stabilizers, diluents, preservatives, solubilizers, liposomes or immunomodulatory agents. Suitable immunomodulatory agents include, but are not limited to, adjuvants, cytokines, polynucleotide encoding cytokines, and agents that facilitate cellular uptake of the PRV-derived immunogenic component.
The PRV immunogenic compositions described herein can also contain an immunostimulatory substance, a so-called adjuvant Adjuvants in general comprise substances that boost the immune response of the host in a non-specific manner. A number of different adjuvants are known in the art. Examples of adjuvants frequently used in fish and shellfish farming are muramyldipeptides, lipopolysaccharides, several glucans and glycans and Carbopol® (a homopolymer). An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review paper by Jan Raa (Reviews in Fisheries Science 4(3): 229-288 (1996)).
The PRV immunogenic compositions described herein may also comprise a so-called “vehicle”. A vehicle is a compound to which the protein adheres, without being covalently bound to it. Such vehicles are e.g. biomicrocapsules, micro-alginates, liposomes and macrosols, all known in the art. A special form of such a vehicle, in which the antigen is partially embedded in the vehicle, is the so-called ISCOM (EP 109.942, EP 180.564, EP 242.380). In addition, the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween. Certain organic solvents such as dimethylsulfoxide also may be employed.
The PRV immunogenic compositions described herein can also be mixed with stabilizers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency. Useful stabilizers are i.e., SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.
When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the immunogenic composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the immunogenic composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and from about 1 to 50% protein or other active ingredient of the present invention.
The PRV immunogenic compositions described herein include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
The PRV immunogenic compositions described herein can also be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
The PRV immunogenic compositions described herein can also be in the form of a complex of the protein(s) or other active ingredient of present invention along with protein or peptide antigens.
The PRV immunogenic compositions described herein can be made suitable for parenteral administration and can include aqueous solutions comprising PRV nucleic acids or polypeptides in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient maybe in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The PRV immunogenic compositions described herein can also be in the form of a liposome in which protein of the present invention is combined, in addition to other acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.
The PRV immunogenic compositions described herein can also be formulated as long acting formulations administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. The compositions may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
Carriers for use with the PRV immunogenic compositions described herein can be a co-solvent systems comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. The proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. The identity of the co-solvent components can also be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
The immunogenic compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with immunogenically compatible counter ions. Such immunogenically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.
Excipients suitable for use in the immunogenic compositions described herein include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
The immunogenic compositions and vaccines described herein can also be multivalent immunogenic compositions that further comprise additional polypeptides or nucleic acid sequences encoding additional polypeptides from other viruses.
The immunogenic compositions and vaccines described herein can also be multivalent immunogenic compositions that further comprise additional polypeptide fragments or nucleic acid sequences encoding additional polypeptide fragments from other viruses.
The immunogenic compositions and vaccines described herein can also be multivalent immunogenic compositions that further comprise additional viruses (e.g. viruses that are either attenuated, killed or otherwise deactivated) or nucleic acid sequences encoding additional viruses (e.g. viruses that are either attenuated, killed or otherwise deactivated).
The immunogenic compositions and vaccines described herein can also comprise fusions proteins, or nucleic acids encoding fusion proteins comprising a PRV polypeptide, or a fragment thereof, and a polypeptide, or a polypeptide fragment from another virus.
Examples of other viruses, viral polypeptides of other viruses or fragments thereof, that can be included in the immunogenic compositions include, but are not limited to, Sleeping disease virus (SDV), or SDV viral polypeptides or fragments thereof; salmon pancreas disease virus (SPDV), or SPDV viral polypeptides or fragments thereof; infectious salmon anemia (ISAV), or ISAV viral polypeptides or fragments thereof; Viral hemorrhagic septicemia virus (VHSV), or VHSV viral polypeptides or fragments thereof; infectious hematopoietic necrosis virus (IHNV), or IHNV viral polypeptides or fragments thereof; infectious pancreatic necrosis virus (IPNV), or IPNV viral polypeptides or fragments thereof; spring viremia of carp (SVC), or SVC viral polypeptides or fragments thereof; channel catfish virus (CCV), or CCV viral polypeptides or fragments thereof; Aeromonas salmonicida, or Aeromonas salmonicida polypeptides or fragments thereof; Renibacterium salmoninarum, or Renibacterium salmoninarum polypeptides or fragments thereof; Moritella viscosis, or Moritella viscosis polypeptides or fragments thereof; Yersiniosis, or Yersiniosis polypeptides or fragments thereof; Pasteurellosis, or Pasteurellosis polypeptides or fragments thereof; Vibro anguillarum, or Vibro anguillarum polypeptides or fragments thereof; Vibrio logei, or Vibrio logei polypeptides or fragments thereof; Vibrio ordalii, or Vibrio ordalii polypeptides or fragments thereof; Vibrio salmonicida, or Vibrio salmonicida polypeptides or fragments thereof; Edwardsiella ictaluri, or Edwardsiella ictaluri polypeptides or fragments thereof; Edwardsiella tarda, or Edwardsiella tarda polypeptides or fragments thereof; Cytophaga columnari, or Cytophaga columnari polypeptides or fragments thereof; or Piscirickettsia salmonis, or Piscirickettsia salmonis polypeptides or fragments thereof.
For example, the cDNA encoding structural protein-1 of infectious salmon anemia virus (ISAV) described in U.S. Pat. No. 6,471,964. ISAV antigens are also disclosed in WO 01/10469. SPDV antigens are disclosed in WO 99/58639. P. salmonis antigens are disclosed in WO 01/68865. Whitespot Virus antigens disclosed in WO 01/09340.
Other viral polypeptides and nucleic acid sequence suitable for use in the immunogenic compositions described herein are discussed in Tucker et al. (2000) “Assessment of DNA vaccine potential for juvenile Japanese flounder Paralichthys olivaceus, through the introduction of reporter genes by particle bombardment and histopathology” Vaccine 19(7-8):801-809; Corbeil et al. (1999) “Evaluation of the protective immunogenicity of the N, P, M, NV, G proteins of infectious hematopoietic necrosis virus in rainbow trout Oncorhynchus mykiss using DNA vaccines” Dis. Aquat. Organ 39(1):29-26; Nusbaum et al. (2002) “Protective immunity induced by DNA vaccination of channel catfish with early and late transcripts of the channel catfish herpes virus (IHV-1)” Vet Immunol. Immunopathol 84(3-4):151-168; Clark et al. (1992) “Developmental expression of surface antigen genes in the parasitic cilate Ichtyophthirius multifiliis” Proc. Natl. Acad. Sci. 89(14):6363-6367; and Sato et al. (2000) “Expression of YAV proteins and vaccination against viral ascites among cultured juvenile yellowtail” Biosci. Biotechnol. Biochem. 64(7):1494-1497. Numerous nucleic acid and amino acid sequences of fish pathogen antigens are known and accessible through the Genbank databases and other sources.
Other additives that are useful in vaccine formulations are known and will be apparent to those of skill in the art.
The following examples illustrate the present invention, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
EXAMPLES
Example 1
Isolation of PRV Fragment
A 200 nt fragment that is approximately 50% homologous at the amino acid level to mammalian Orthoreoviruses was obtained through high throughput sequencing of samples obtained from farmed salmon with HSMI in Norway. Quantitative PCR assays of muscle tissue from salmon with HSMI and normal salmon reveals a higher viral load in salmon with HSMI.
Example 2
Heart and Skeletal Muscle Inflammation of Farmed Salmon is Associated with Infection with a Novel Reovirus
RNA extracted from heart of a salmon with experimentally induced HMSI was pyrosequenced (Margulies, M. et al., Nature 437, 376-380 (2005)) yielding 106,073 reads ranging in size up to 598 nucleotide (average=349.7, SD=149.5). Although database alignment analysis at the nucleotide level revealed no evidence of infection, the predicted amino acid sequence of one 265 nucleotide read was 49% similar to the core-spike protein λ2 of Mammalian orthoreovirus 3 (AF378009). A real time PCR assay based on this sequence was used to test for the presence of the candidate virus in RNA extracts of heart and serum obtained from salmon with HSMI in association with spontaneous outbreaks (n=20) or experimental infection (n=20), and in non-infected control fish (n=20). All samples from salmon with HSMI contained the candidate sequences. No sequences were found in the control salmon without HSMI.
The HSMI serum sample with the highest genetic load by PCR (3.0×106 genome copies/μ1) was selected for additional pyrosequencing yielding 120,705 reads. A suite of bioinformatic tools was used to identify viral sequences. In the first phase of analysis, BLASTN and BLASTX (Altschul et al., J Mol Biol 215, 403-410 (1990)) detected 1.5% and 53.9% of the predicted viral genome, respectively, enabling identification of segments L1, L2, L3, M1, M2 and M3 (FIG. 1). Implementation of FASTX (Pearson et al., Genomics 46, 24-36(1997)) yielded an additional 5.5% of the genome and detected motifs in the S1 segment as well as additional sequences in the L2 and M3 segments. Frequency Analysis of Sequence Data (FASD) (Trifonov et al, (submitted)), a program that predicts taxonomy based on nucleotide frequency and order rather than sequence alignment, detected new sequences representing the S1, S2, S3 and S4 segments (FIG. 1) that comprised an additional 11.8% of the final viral genome assembly. In total, approximately 17 kilobases of sequence (72.8% of the genome) was obtained by pyrosequencing (FIG. 1). Gaps between fragments and the termini of gene segments were completed by PCR cloning. All sequence was verified by classical dideoxy sequencing by using primers designed along the draft sequence.
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). Reoviruses are non-enveloped icosahedral viruses with double-stranded RNA genomes comprising 10-12 segments. Twelve genera are defined based on host range, number of genome segments, G/C content, and antigenic relationships. A phylogenetic tree constructed using L gene segment sequences of known reoviruses indicate that PRV represents a distinct genetic lineage branching off the root of the aquareovirus and orthoreovirus genera, viruses of fish and shellfish, reptiles, birds and mammals (FIG. 2). Analysis of all ten PRV gene segments confirmed the divergence of PRV sequence with respect to other Reoviruses (FIGS. 5 to 12). All PRV gene segments contained the 3′ terminal nucleotides (UCAUC-3′) found in orthoreoviruses and aquareoviruses (Attoui et al., J Gen Virol 83, 1941-1951 (2002)); however, the 5′ terminal nucleotides (5′-GAUAAA/U) were unique.
The orthoreoviruses have polycistronic segments in either S1 or S4. Whereas aquareovirus species C are polycistronic in the S7 (the orthoreovirus S1 homolog), the other aquareovirus species are not (Attoui et al., J Gen Virol 83, 1941-1951 (2002)). PRV has a putative open reading frame (ORF) in the 5′-end of S2 (71 aa, pI=8.8, 8 kDa), and a putative ORF in 5′-end of S1 (124 aa, pI=4.8, 13 kDa). Although homologues of the λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σNS sequences of PRV are found in orthoreoviruses and aquareoviruses, the σ1 and σ3 sequences and the small putative open reading frames observed in S2 and S1 appear distinctive. The structure of the latter is similar to a fusion-associated small transmembrane (FAST) reovirus protein (Shmulevitz et al., EMBO J 19, 902-912 (2000)) (FIG. 13). Reovirus FAST proteins are nonstructural, single-pass membrane proteins that induce cell-cell fusion and syncytium formation (Shmulevitz et al., EMBO J 19, 902-912 (2000)). Taken together these data provide compelling evidence that PRV is the prototype of a new reovirus genus equally distant to the orthoreovirus and aquareovirus genera.
The prevalence of PRV infection in farmed and wild salmon was examined using real time PCR assays targeting genome segments L1, L2, M3 and S4. Levels of viral RNA were quantitated using an MGB assay against L1 wherein results were normalized to elongation factor 1A (EF1A) using the formula by Pfaffl (Pfaffl et al., Nucleic Acids Res 29, e45 (2001)). Heart and kidney samples from 29 salmon representing three different HSMI outbreaks were studied (Table 1) and 10 samples from healthy farmed fish. Twenty-eight of the 29 (96.5%) known HSMI samples and none of the 10 (0%) healthy salmon samples were positive as defined by L1/EF1A gene log ratio ≧5.00. Only one of 29 HSMI samples was negative; this sample originated from a salmon net harboring fish in the early phase of HSMI, prior to the onset of fish mortality (FIG. 3). In fish with signs of severe disease, including abnormal swimming behavior, anorexia and histologic evidence of pancarditis and myositis (Kongtorp et al., J Fish Dis 29, 233-244 (2006)), the median adjusted L1/EF1A gene log ratio was 10.36 (IQR, 0.94). The L1/EF1A gene log ratio was correlated not only with the presence or absence of HSMI, but also, with severity of disease at the time of sampling. The log ratios were lowest in healthy farmed salmon (log ratio range, −0.23 to 3.89; n=10), higher in salmon collected in the early phase of an HSMI outbreak (range, 4.34 to 7.66; n=10), and highest in salmon obtained at the peak of an HSMI outbreak (range, 8.52 to 11.90; n=10). To study the prevalence and relative levels of PRV in healthy wild salmon from different geographic locations, 66 samples obtained from nine coastal rivers in Norway were tested. PRV was detected in only sixteen of these samples (24.2%). Two of these sixteen samples were positive by the cutoff established for farmed salmon with relative log ratios of 6.70 and 7.58; the other fourteen had L1/EF1A log ratios well below the 5.00 cutoff (range, −0.20 to 4.57). No PRV transcripts were detected in any of the remaining wild salmon samples (n=50).







TABLE 1







Viral burden data.


















Outbreak


log







group/

L1/EF1A
L1/EF1A

Positive/


Sample
Fish
Disease
disease

gene ratio
gene
Virus
negative


ID
type
status
phase
Tissue
(adjusted)a
ratiob
detectionc
(min = 5.00)d


















408-1
Farmed
HSMI
Farmed
Heart/
3.3E+08
8.52
+
Positive





HSMI-
kidney





peak





phase


408-2
Farmed
HSMI
Farmed
Heart/
1.1E+10
10.06
+
Positive





HSMI-
kidney





peak





phase


408-3
Farmed
HSMI
Farmed
Heart/
6.4E+09
9.80
+
Positive





HSMI-
kidney





peak





phase


408-4
Farmed
HSMI
Farmed
Heart/
1.8E+09
9.26
+
Positive





HSMI-
kidney





peak





phase


408-5
Farmed
HSMI
Farmed
Heart/
5.5E+10
10.74
+
Positive





HSMI-
kidney





peak





phase


408-6
Farmed
HSMI
Farmed
Heart/
7.1E+09
9.85
+
Positive





HSMI-
kidney





peak





phase


408-7
Farmed
HSMI
Farmed
Heart/
6.9E+10
10.84
+
Positive





HSMI-
kidney





peak





phase


408-8
Farmed
HSMI
Farmed
Heart/
8.0E+11
11.90
+
Positive





HSMI-
kidney





peak





phase


408-9
Farmed
HSMI
Farmed
Heart/
5.2E+10
10.71
+
Positive





HSMI-
kidney





peak





phase


408-10
Farmed
HSMI
Farmed
Heart/
4.6E+10
10.67
+
Positive





HSMI-
kidney





peak





phase


SK300
Farmed
HSMI
Farmed
Heart/
2.8E+10
10.45
+
Positive





HSMI
kidney





outbreak


SK301
Farmed
HSMI
Farmed
Heart/
1.6E+09
9.20
+
Positive





HSMI
kidney





outbreak


SK302
Farmed
HSMI
Farmed
Heart/
1.9E+09
9.28
+
Positive





HSMI
kidney





outbreak


SK303
Farmed
HSMI
Farmed
Heart/
1.9E+07
7.28
+
Positive





HSMI
kidney





outbreak


SK304
Farmed
HSMI
Farmed
Heart/
2.7E+08
8.44
+
Positive





HSMI
kidney





outbreak


SK305
Farmed
HSMI
Farmed
Heart/
2.6E+07
7.42
+
Positive





HSMI
kidney





outbreak


SK306
Farmed
HSMI
Farmed
Heart/
5.6E+08
8.75
+
Positive





HSMI
kidney





outbreak


SK307
Farmed
HSMI
Farmed
Heart/
5.9E+08
8.77
+
Positive





HSMI
kidney





outbreak


SK308
Farmed
HSMI
Farmed
Heart/
2.0E+09
9.29
+
Positive





HSMI
kidney





outbreak


562-1
Farmed
HSMI
Farmed
Heart/
1.4E+06
6.14
+
Positive





HSMI-
kidney





early





phase


562-2
Farmed
HSMI
Farmed
Heart/
1.5E+06
6.16
+
Positive





HSMI-
kidney





early





phase


562-3
Farmed
HSMI
Farmed
Heart/
1.3E+06
6.10
+
Positive





HSMI-
kidney





early





phase


562-4
Farmed
HSMI
Farmed
Heart/
9.6E+05
5.98
+
Positive





HSMI-
kidney





early





phase


562-5
Farmed
HSMI
Farmed
Heart/
2.2E+04
4.34
+
Negative





HSMI-
kidney





early





phase


562-6
Farmed
HSMI
Farmed
Heart/
1.6E+07
7.22
+
Positive





HSMI-
kidney





early





phase


562-7
Farmed
HSMI
Farmed
Heart/
4.6E+07
7.66
+
Positive





HSMI-
kidney





early





phase


562-8
Farmed
HSMI
Farmed
Heart/
1.5E+05
5.18
+
Positive





HSMI-
kidney





early





phase


562-9
Farmed
HSMI
Farmed
Heart/
2.8E+05
5.44
+
Positive





HSMI-
kidney





early





phase


 562-10
Farmed
HSMI
Farmed
Heart/
1.2E+07
7.07
+
Positive





HSMI-
kidney





early





phase


PD
Farmed
Healthy
Farmed
Heart/
7.6E+02
2.88
+
Negative


3511


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
1.2E+02
2.07
+
Negative


3512


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
2.5E+03
3.41
+
Negative


3513


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
7.9E+03
3.90
+
Negative


3514


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
4.8E+03
3.68
+
Negative


3515


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
4.2E+01
1.62
+
Negative


3516


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
4.5E+03
3.65
+
Negative


3517


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
5.8E−01
−0.23
+
Negative


3518


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
1.1E+03
3.02
+
Negative


3519


healthy
kidney


PD
Farmed
Healthy
Farmed
Heart/
2.1E+03
3.32
+
Negative


3520


healthy
kidney


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


350


healthy


SF/08
Wild
Healthy
Wild
Heart
4.5E+02
2.66
+
Negative


351


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


353


healthy


SF/08
Wild
Healthy
Wild
Heart
5.0E+02
2.7
+
Negative


354


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


315


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


316


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


319


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


321


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


325


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


332


healthy


SF/08
Wild
Healthy
Wild
Heart
6.3E−01
−0.2
+
Negative


338


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


48


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


50


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


53


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


56


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


60


healthy


SF/08
Wild
Healthy
Wild
Heart
5.0E+03
3.7
+
Negative


61


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


62


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


63


healthy


SF/08
Wild
Healthy
Wild
Heart
3.1E+03
3.49
+
Negative


64


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


432


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


438


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


440


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


442


healthy


SF/08
Wild
Healthy
Wild
Heart
5.1E+02
2.71
+
Negative


444


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


446


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


447


healthy


SF/08
Wild
Healthy
Wild
Heart
3.7E+04
4.57
+
Negative


452


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


453


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


463


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


464


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


477


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


491


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


497


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


508


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


511


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


517


healthy


SF/08
Wild
Healthy
Wild
Heart
1.7E+01
1.23
+
Negative


518


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


519


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


522


healthy


SF/08
Wild
Healthy
Wild
Heart
5.0E+06
6.7
+
Positive


198


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


200


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


201


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


205


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


206


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


207


healthy


SF/08
Wild
Healthy
Wild
Heart
3.8E+07
7.58
+
Positive


208


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


209


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


210


healthy


SF/08
Wild
Healthy
Wild
Heart
—
—
−
Negative


211


healthy


  1-13
Wild
Healthy
Wild
Heart
1.2E+01
1.08
+
Negative





healthy


  1-14
Wild
Healthy
Wild
Heart
—
—
−
Negative





healthy


  1-21
Wild
Healthy
Wild
Heart
—
—
−
Negative





healthy


  1-22
Wild
Healthy
Wild
Heart
—
—
−
Negative





healthy


  1-23
Wild
Healthy
Wild
Heart
—
—
−
Negative





healthy


  1-24
Wild
Healthy
Wild
Heart
1.7E+00
0.24
+
Negative





healthy


1H
Wild
Healthy
Wild
Heart
5.4E+01
1.73
+
Negative





healthy


2H
Wild
Healthy
Wild
Heart
—
—
−
Negative





healthy


3H
Wild
Healthy
Wild
Heart
—
—
−
Negative





healthy


1M
Wild
Healthy
Wild
Muscle
4.0E+01
1.6
+
Negative





healthy


2M
Wild
Healthy
Wild
Muscle
—
—
−
Negative





healthy


3M
Wild
Healthy
Wild
Muscle
1.7E+02
2.23
+
Negative





healthy


1Mi
Wild
Healthy
Wild
Spleen
—
—
−
Negative





healthy


2Mi
Wild
Healthy
Wild
Spleen
—
—
−
Negative





healthy


3Mi
Wild
Healthy
Wild
Spleen
—
—
−
Negative





healthy


521-6
Wild
Healthy
Wild
Various
2.7E+00
0.42
+
Negative





healthy
organs





a= Ratio of virus burden (quantitated through the L1 viral gene), normalized using a salmon housekeeping gene (EF1A) and adjusted by a factor of 108.


b= Log transformation of the adjusted ratio L1/EF1A.


c= Virus detection by real time RT-PCR.


d= For statistical analyses, samples were considered positive whenever the adjusted log ratio was higher than 5.00






The anatomic distribution of PRV in relation to pathology was tested through in situ hybridization using probes to L2 gene RNA. PRV RNA was distributed throughout the myocardium and endocardium of salmon with HSMI (FIG. 4A, 4B) but not detected in normal salmon or salmon infected with salmon pancreas disease virus (FIG. 4C, 4D)
Implication of a microbe in a disease via Koch's postulate requires demonstration that an agent is specific for that disease, and that disease can be reproduced in a naïve host by inoculation with the agent propagated in culture following isolation from an affected host. Although fulfillment of this postulate is compelling evidence of causation the criteria are unduly stringent. Some agents cannot be cultured. Additionally, genetic and other factors may contribute to pathogenesis. PRV has not been cultured. Furthermore, PRV has been found in farmed fish that do not show clinical signs of HSMI. Moreover, PRV has been also detected in low quantities in wild Atlantic salmon. Nonetheless, the tissue distribution and load of PRV are correlated with disease in naturally and experimentally infected salmon. Analogies between commercial poultry production and Atlantic salmon aquaculture may be informative Reoviruses are also implicated in numerous diseases of poultry, including enteritis, myocarditis, and hepatitis (Jones, Rev Sci Tech 19, 614-625 (2000)). Both poultry production and aquaculture confine animals at high density in conditions that are conducive to transmission of infectious agents and may reduce resistance to disease by induction of stress.
Unlike terrestrial animal farming, where contact between domestic and free ranging wild animals of the same or closely related species is easily monitored and controlled, ocean based aquaculture is an open system wherein farmed fish may incubate and transmit infectious agents to already diminishing stocks of wild fish. PRV will be isolated in cell culture and prevention or modification of the disease will be performed disease through use of specific drugs or vaccines. Nonetheless, the results described herein show that a causal relationship can exists, measures to control PRV can be undertaken because PRV threatens domestic salmon production and also has the potential for transmission to wild salmon populations.
Example 3
PRV Identification and Sequencing
HSMI was experimentally-induced in normal Atlantic salmon by inoculation with heart and kidney extracts from fish with HSMI or cohabitation with fish with HSMI. RNA extracted from heart tissue from Atlantic salmon with experimentally-induced HSMI was used as template for high throughput pyrosequencing. Sequences were analyzed using a suite of bioinformatic applications available at the GreenePortal website (http://tako.cpmc.columbia edu/Tools), including FASD, a method whereby the statistical distribution of oligonucleotide frequencies within an unknown sequence set is compared to frequencies calculated for known sequence sets. Seven of ten segments of a novel reovirus, piscine reovirus (PRV), were identified using alignment and a motif-based program; three additional segments were identified using FASD. Quantitative real time PCR assays of samples from fish collected during outbreaks of HSMI and from fish with experimentally-induced HSMI confirmed association between PRV and HSMI. In situ hybridization confirmed the presence of PRV sequences in heart of fish with HSMI.
Identification of PRV by high-throughput sequencing Healthy Atlantic salmon produced at an experimental facility (VESO, Vikan; Namsos, Norway), with an average weight of 50 g were inoculated with cardiac tissue from field outbreaks of HSMI and served as donors for material for the high-throughput sequencing. Non-inoculated fish served as negative controls (Kongtorp and Taksdal, J Fish Dis 32, 253-262 (2009)). Three heart muscle biopsies were diluted 1:10 in HBSS, filtrated through a 0.22 μm filter and inactivated in TRIzol LS reagent. Several serum samples were inactivated directly in TRIzol LS. Total RNA extracts were treated with DNase I and cDNA generated by using the Superscript II system for reverse transcription primed by random octamers that were linked to an arbitrary defined 17-mer primer sequence (Palacios et al., Emerg Infect Dis 13, 73-81 (2007)). The resulting cDNA was treated with RNase H and then randomly amplified by the polymerase chain reaction (PCR); applying a 9:1 mixture of a primer corresponding to the defined 17-mer sequence and the random octamer-linked 17-mer primer, respectively (Palacios et al., Emerg Infect Dis 13, 73-81 (2007)). Products >70 base pairs (bp) were selected by column purification and ligated to specific linkers for sequencing on the 454 Genome Sequencer FLX without fragmentation of the cDNA (Margulies, M. et al., Nature 437, 376-380 (2005); Palacios et al. N Engl J Med 358, 991-998 (2008); Cox-Foster et al., Science 318, 283-287 (2007)).
Removal of primer sequences, redundancy filtering, and sequence assembly were performed with software programs accessible through the analysis applications at the GreenePortal website (http://156.145.83 115/Tools). When traditional BLASTN, BLASTX and FASTX analysis failed to identify the origin of the sequence read, FASD was applied (Trifonov et al, (submitted)), a novel method based on the statistical distribution of oligonucleotide frequencies. The probability of a given segment to belong to a class of viruses is computed from their distribution of oligonucleotide frequencies in comparison with the calculated for other segments. A statistic measure was developed to assess the significance of the relation between segments. The p-value estimates the likelihood that an oligonucleotide distribution is derived from a different one. Thus, highly related distributions present a high p-value.
Conventional PCRs were performed with HotStar polymerase on PTC-200 thermocyclers an enzyme activation step of 5 min at 95° C. was followed by 45 cycles of denaturation at 95° C. for 1 min, annealing at 55° C. for 1 min, and extension at 72° C. for 1 to 3 min depending on the expected amplicon size. Amplification products were run on 1% agarose gels, purified and directly sequenced in both directions with ABI PRISM Big Dye Terminator 1.1 Cycle Sequencing kits on ABI PRISM 3700 DNA Analyzers.
Example 4
Sequence Analyses
Programs of the Geneious package (Biomatters, New Zealand) were used for sequence assembly and analysis. Sequences were downloaded from GenBank and aligned using the ClustalX (Thompson et al., Curr Protoc Bioinformatics Chapter 2, Unit 23 (2002)) implementation on the MEGA software (Tamura et al., Mol Biol Evol 24, 1596-1599 (2007)). The amino acid alignments obtained were further refined using T-Coffee (Notredame et al., J Mol Biol 302, 205-217 (2000)) to incorporate protein structure data on the alignment. To evaluate the robustness of the approach, the ability to find and align motifs previously identified as conserved among Reoviridae was used as a marker. Phylogenetic analysis were performed using p-distance as model of amino acid substitution as accepted by ICTV for analysis of the Reoviridae family. MEGA was used to produce phylogenetic trees, reconstructed through the Neighbor Joining (NJ) method.
The statistical significance of a particular tree topology was evaluated by bootstrap resampling of the sequences 1000 times. Bayesian phylogenetic analyses of the sequence differences among segments λ1, λ2, λ3, μ1, μ2, μ3, σ2 and σ3 (σ1 and σNS of aquareovirus and orthoreovirus had different genomic organizations) were conducted using the BEAST, BEAUti and Tracer analysis software packages.
Preliminary analyses were run for 10,000,000 generations with the Dayhoff amino acid substitution model to select the clock and demographic models most appropriate for each ORF. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was chosen for all datasets. Final data analyses included MCMC chain lengths of 5,000,000-30,000,000 generations, with sampling every 1000 states (FIGS. 5-12).
Example 5
Real Time PCR
Quantitative assays were established based upon virus specific sequences obtained from the high throughput sequencing for several reovirus segments. Six different realtime assays were designed targeting genome fragment L1, L2 and M3 (SYBR green) as well as L1 and S4 (MGB assays) (See Table 2 for a list of the primers). Samples from different organs from experimentally infected fish were positive while samples from non-infected control fish were negative. For further screening, the real-time PCR for segment L1 was performed using the QIAGEN OneStep kit. Six μl of template RNA were denatured (95° C./5 min). Reactions were performed using the following concentrations: 400 nM primer, 300 nM probe and 1.25 mM MgCl2. Amplifications were done in a Stratagene Mx3005P real-time PCR machine (Stratagene) with the following cycle parameters: 30 min at 50° C. (reverse transcription), 15 min at 94° C. (RT inactivation and PCR polymerase activation), 45 cycles of 94° C./15 sec, 54° C./30 sec and 72° C./15 sec. Standard curves were created using RNA pooled from three fish with high viral loads. Standard curves were made in duplicates for both the MGB assay and the EF1A assay (Olsvik et al., BMC Mol Biol 6, 21 (2005)) and relative viral RNA loads for field samples were calculated by using normalization against EF1A.







TABLE 2 







Primers for realtime assays for targeting genome fragment L1, L2 and


M3 (SYBR green) as well as L1 and S4.












Assay
Target




Primer name
type
segment
Sequence (5′-3′)
SEQ ID NO














AqureoGT70F
SYBR
L2
AGGATGTATGCCACTAGCTCC
SEQ ID NO: 11



green
(1577-1561)




AqureoGT70R
SYBR
L2
GCTGGTAACTGGCTTACTGCTAAT
SEQ ID NO: 12



green
 1513-1536)




 


AquareoHC86F
SYBR
L1
ATGTCACAACTTGAGTCAGTTCC
SEQ ID NO: 13



green
(3832-3810)




AquareoHC86R
SYBR
L1
GATACAGCTACCCAACATGATTGA
SEQ ID NO: 14



green
(3747-3770)




 


AquareoNS86F
SYBR
M3
TCAGTGCGGGGAACTCTAGTGGCA
SEQ ID NO: 15



green
(2119-2096)




AquareoNS86R
SYBR
M3
GACGACCTTGAACGCACGAGCGTG
SEQ ID NO: 16



green
(2025-2048)




 


Salmon_Reo_F
SYBR
L2
TGCTGGCGATGATCTTGGAGTATGC
SEQ ID NO: 17



green
(1767-1792)




Salmon_Reo_R
SYBR
L2
ACACCATCAGTGAACTTAGGAGCAACA
SEQ ID NO: 18



green
(1908-1935)




 


L1_2671F
MGB
L1
TGCTAACACTCCAGGAGTCATTG
SEQ ID NO: 19



assay
(3219-3241)




L1_2729R
MGB
L1
TGAATCCGCTGCAGATGAGTA
SEQ ID NO: 20



assay
(3277-3257)




L1 MGB probe
MGB
L1
FAM - CGCCGGTAGCTCT -
SEQ ID NO: 21



assay
(3243-3256)
MGBNFQ



 


S4_F1
MGB
S1
ACAGTCGCGGTTCAAACGA
SEQ ID NO: 22



assay
(399-417)




S4_R2
MGB
S1
AAGGCGTCGCTTAGCTTCAA
SEQ ID NO: 23



assay
(460-441)




S4 MGB probe
MGB
S1
FAM - AGACCAGACAGACGC -
SEQ ID NO: 24



assay
(419-433)
MGBNFQ



 


ELAF
TAQMAN
Elongation
CCACAGACAAGCCCCTTCGT
SEQ ID NO: 25


ELAR
TAQMAN
factor A
CCTTCAGGGTTCCAGTCTCCA
SEQ ID NO: 26


ELA probe
TAQMAN

FAM -
SEQ ID NO: 27





AGGTACAGTTCCAATACCACCGATTTT






GTAAACG - TAMRA









Example 6
In Situ Hybridization
In situ hybridization was performed in compliance with the protocol from GeneDetect (Auckland, New Zealand) with some modifications using LNA probes targeting L2. Sections were permeabilized using 40 μml-1 Proteinase K in TE buffer at 37° C. for 15 min followed by hybridization with a mixture of two 5′ and 3′ double DIG labeled LNA probes (5′-CACCATCAGTGAACTTAGGAGCAAC-3′ and 5′-CATACTCCAAGATCATCGCCAGCA-3′) (SEQ ID NO: 28 and SEQ ID NO: 41, respectively) (250 nM each) for 18 hours at 50° C. Stringency washes were carried out at 60° C.
Sections were incubated with a mouse monoclonal anti-DIG-HRP overnight at 4° C. and stained using a Tyramide Signal Amplification System (Perkin Elmer, MA, USA) according to the manufacturer's protocol. Sections were counterstained with Meyer's hematoxylin solution. Negative controls included were samples from non-infected fish from experimental trial, head kidney samples from non-infected fish as a source of immune cells, salmon with pancreatic disease (a differential diagnosis to HSMI), and samples from material sent for diagnostics at random.
Example 7
Statistical Analysis
StatView version 5.0.1 software for Windows (SAS Institute, Cary, N.C., USA) was used for all statistical analyses. Samples without detectable L1 viral gene transcripts were excluded from statistical analysis. Log transformations were performed for all other samples after calculating L1/EF1A ratios (adjusted by a factor of 108). Log-transformed data were retained to facilitate graphical display of group differences, though distributions were not normalized by this method; thus, nonparametric analytic approaches were employed (Mann-Whitney U-test for comparison of healthy and HSMI fish; Kruskal-Wallis for comparisons of healthy and early, middle and peak phase HSMI fish). For all tests, statistical significance was assumed where p<0.05.
Example 8
Propagation of Virus In Cell Culture
Syncytium formation and vacuolization can be observed after infecting epithelioma paplosum cyprini (EPC) cells and fat head minnow (FHM) cells with tissue homogenate from HSMI diagnosed fish, however the cytopathic effect (CPE) is rarely seen after 2 to 4 passages.
Example 9
Challenge of Atlantic Salmon
Experimental challenge by injecting Atlantic salmon with material from HSMI diagnosed fish shows pathological changes consistent with HSMI.
Example 10
Electron Microscopy
Virus-like particles of 60 to 80 nm diameter are been observed in necrotic cardiomyocytes diagnosed with HSMI. Chloroform sensitivity analysis shows that PRV belongs to the Reoviridae family, which is a family of naked viruses.
Example 11
Screening of Heart Samples from Experimental Challenge
Heart samples were screened by RT-qPCR for quantification of virus after challenge of Atlantic salmon with tissue homogenate from HSMI diagnosed fish. 10 weeks post challenge (wpc), 4 of 5 fish were positive for the virus (Table 3). The results are consistent with the pathological findings.







TABLE 3







Quantification of virus in heart samples after challenge.









Wpc

















0
1
2
3
4
5
6
8
10




















Positive
0/5
1/5
1/5
0/5
0/5
0/5
0/5
1/5
4/5


(Ct)

(40)
(38)




(39)
(21-36)





Wpc = weeks post challenge.






Example 12
Immunization of Rabbits
The open reading frame (ORF), minus the 126 first nucleotides, of the M2 genomic segment (SEQ ID NO: 5) encoding the μ1 protein was cloned in the pET100 plasmid and expressed as His-tag fusion protein in E. coli, purified. The μ1 protein is posttranscriptionally cleaved into μ1c in mammalian orthoreovirus in a process wherein 42 aa are removed from the N-terminus of μ1. The protein was used for immunization of a rabbit to obtain polyclonal, μ1C-specific antiserum. The antiserum recognizes the μ1c protein as found in Western blots of E. coli His-tag fusion protein and different negative controls (FIG. 20). The antiserum recognizes PRV, as has been shown in immunohistochemistry of hearts of fish with HSMI.
The open reading frame (ORF), from nucleotide 29-1018 of the 51 genomic segment (SEQ ID NO: 2) encoding the σ3 protein (330 amino acids long) (SEQ ID NO: 39) was cloned in the pET101 plasmid and expressed as His-tag fusion protein in E. coli, purified and used for immunization for a rabbit to obtain polyclonal, σ3-specific antiserum. The antiserum recognizes the s3 protein as found in western blots of E. coli His-tag fusion protein and different negative controls. The antiserum recognizes native PRV, as has been shown in immunohistochemistry of heart of fish with HSMI.
The open reading frame (ORF), from the nucleotide 22-1281 of the S2 genomic segment (SEQ ID NO: 2) encoding the σ1 protein (420 amino acids long) (SEQ ID NO: 35) was cloned in the pET101 plasmid and expressed as a His-tag fusion protein in E. Coli, purified and used for immunization of a rabbit to obtain polyclonal, σ2-specific antiserum. The antiserum recognizes the σ1 protein as found in western blots of E. coli His-tag fusion protein (FIG. 21) and in immunohistochemistry of hearts of fish with HSMO.
The open reading frame (ORF), from nucleotide 39-983 of the S4 genomic segment (SEQ ID NO: 3) encoding the σ2 protein (SEQ ID NO: 38) (315 amino acids long) was cloned in the pET100 plasmid and expressed in E. coli. Purification of protein is ongoing.
Peptides were synthesized form the amino acid sequences of assumed antigenic region from the fusion-associated small transmembrane protein (FAST) protein (SEQ ID NO: 40) encoded by S1 (SEQ ID NO: 2) (nucleotide 108-479, +1 frame relative to the ORF of σ3) and was used for immunization of a rabbit to obtain polyclonal FAST-specific antiserum. Currently it is being tested by immunohistochemistry of hearts of fish with HSMI of the antiserum recognizes PRV infected cells.
Rabbits were immunized (3rd booster) with recombinant proteins expressed in E. coli. The outer capsid proteins sigma-1 (SEQ ID NO: 35), sigma-3 (SEQ ID NO: 37) and mu-1C (SEQ ID NO: 33) were expressed and injected, in addition to a synthetic peptide of the FAST protein of S1 (SEQ ID NO: 40). Specific antibodies targeting the FAST protein can increase chances of culturing the virus, as the FAST protein is involved in syncytium formation.
The sera raised against the μ1, σ3 and putative σ2 proteins all give positive signals in immunohistochemistry of hearts from salmon with HSMI. The serum against the μ1 protein works best and gives a good signal to noise ratio in immunohistochemistry.







TABLE 4







Annotation of ORFs proteins. Based on in silico analysis.













Putative Function of


Genomic Segment of

PRV Protein
PRV Compared to MRV,


PRV
PRV Proteins
SEQ ID NO
ARV and GCRV





L3 (SEQ ID NO: 9)
λ3, 144.3 kDa, 1286 aa
SEQ ID NO: 31
RNA-dependent RNA





polymerase


L2 (SEQ ID NO: 10)
λ2, 143.7 kDa, 1290 aa
SEQ ID NO: 30
Guanylyltransferase,





methyltransferase


L1 (SEQ ID NO: 8)
λ1, 141.1 kDa, 1282 aa
SEQ ID NO: 29
Helicase, NTPase


M1 (SEQ ID NO: 6)
μ2, 86.1 kDa, 760 aa
SEQ ID NO: 33
NTPase


M2 (SEQ ID NO: 5)
μ1, 74.2 kDa, 687 aa
SEQ ID NO: 32
Outer capsid


M3 (SEQ ID NO: 7)
μNS, 83.5 kDa, 752 aa
SEQ ID NO: 34
dsRNA binding


S2 (SEQ ID NO: 4)
σ1, 45.9 kDA, 420 aa
SEQ ID NO: 35
Inner capsid



(S2 ORF 1)


S2 (SEQ ID NO: 4)
σ1 s, 10.9 kDa, 71aa
SEQ ID NO: 36
Inner capsid



(S2 ORF 2)


S4 (SEQ ID NO: 3)
σ2, 34.6 kDa, 315 aa
SEQ ID NO: 38
Cell attachment, primary





serotype determinant


S3 (SEQ ID NO: 1)
σNS, 39.1 kDa, 354 aa
SEQ ID NO: 37
dsRNA binding


S1 (SEQ ID NO: 2)
σ3 37.0 kDa, 330 aa
SEQ ID NO: 39
Zinc mettaloprotein



(S1 ORF 1)


S1 (SEQ ID NO: 2)
FAST 13.0 kDa, 124 aa
SEQ ID NO: 40
FAST protein



(S1 ORF 2)









Example 13
Virus Characterization and Virulence Studies
PRV virus segments were cloned and expressed in insect and fish cell lines to examine potential virulence factors virus characterization and virulence studies. The hemagglutinating properties of the virus will also be tested and samples from geographically distant areas will be sequenced to study potential differences and strain variations.
Example 14
Screening of Wild Fish and Fertilized Eggs
Material from the National Gene Bank will be screened for presence of the virus in wild salmon populations and examined for possible vertical transfer of virus.
Example 15
Screening of Brood Stocks
Material for screening of brood stocks kept under different conditions can be obtained, inter alia, from one or more commercial breeding companies.
REFERENCES
Kongtorp, R. T., Kjerstad, A., Taksdal, T., Guttvik, A. & Falk, K. Heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L: a new infectious disease. J Fish Dis 27, 351-358, doi:10.1111/j.1365-2761.2004.00549.xJFD549 [pii] (2004).
Ferguson, H. W., Kongtorp, R. T., Taksdal, T., Graham, D. & Falk, K. An outbreak of disease resembling heart and skeletal muscle inflammation in Scottish farmed salmon, Salmo salar L., with observations on myocardial regeneration. J Fish Dis 28, 119-123, doi:JFD602 [pii] 10.1111/j.1365-2761.2004.00602.x (2005).
Kongtorp, R. T., Taksdal, T. & Lyngoy, A. Pathology of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar. Dis Aquat Organ 59, 217-224 (2004).
Watanabe, K. et al. Virus-like particles associated with heart and skeletal muscle inflammation (HSMI). Dis Aquat Organ 70, 183-192 (2006).
Margulies, M. et al. Genome sequencing in microfabricated high-density picoliter reactors. Nature 437, 376-380 (2005).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J Mol Biol 215, 403-410, doi:10.1006/jmbi.1990.9999 S0022283680799990 [pii] (1990).
Pearson, W. R., Wood, T., Zhang, Z. & Miller, W. Comparison of DNA sequences with protein sequences. Genomics 46, 24-36, doi:S0888-7543(97)94995-8 [pii] 10.1006/geno.1997.4995 (1997).
Trifonov, V. & Rabadan, R. Frequency analysis techniques for discovery of novel microorganisms. mBio (submitted).
Attoui, H. et al. Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae). J Gen Virol 83, 1941-1951 (2002).
Shmulevitz, M. & Duncan, R. A new class of fusion-associated small transmembrane (FAST) proteins encoded by the non-enveloped fusogenic reoviruses. EMBO J 19, 902-912, doi:10.1093/emboj/19.5.902 (2000).
Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, e45 (2001).
Kongtorp, R. T., Halse, M., Taksdal, T. & Falk, K. Longitudinal study of a natural outbreak of heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L. J Fish Dis 29, 233-244, doi:JFD710 [pii] 10.1111/j.1365-2761.2006.00710.x (2006).
Jones, R. C. Avian reovirus infections. Rev Sci Tech 19, 614-625 (2000).
Thompson, J. D., Gibson, T. J. & Higgins, D. G. Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics Chapter 2, Unit 23, doi:10.1002/0471250953.bi0203s00 (2002).
Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599, doi:msm092 [pii] 10.1093/molbev/msm092 (2007).
Notredame, C., Higgins, D. G. & Heringa, J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol 302, 205-217, doi:10.1006/jmbi.2000.4042 S0022-2836(00)94042-7 [pii] (2000).
Mertens, P., Attoui, H., Duncan, R. & Dermody, T. Family Reoviridae. 447-454 (Elsevier Academic Press, 2005).
Kongtorp, R. T. & Taksdal, T. Studies with experimental transmission of heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L. J Fish Dis 32, 253-262, doi:JFD983 [pii] 10.1111/j.1365-2761.2008.00983.x (2009).
Palacios, G. et al. Panmicrobial oligonucleotide array for diagnosis of infectious diseases. Emerg Infect Dis 13, 73-81 (2007).
Palacios, G. et al. A new arenavirus in a cluster of fatal transplant-associated diseases. N Engl J Med 358, 991-998, doi:NEJMoa073785 [pii] 10.1056/NEJMoa073785 (2008).
Cox-Foster, D. L. et al. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318, 283-287, doi:1146498 [pii] 10.1126/science.1146498 (2007).
Olsvik, P. A., Lie, K. K., Jordal, A. E., Nilsen, T. O. & Hordvik, I. Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol Biol 6, 21, doi:1471-2199-6-21 [pii] 10.1186/1471-2199-6-21 (2005).
Kongtorp R. T., Taksdal T. & Lyngøy A. (2004b) Pathology of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar. Diseases of Aquatic Organisms 59, 217-224.
Kongtorp R. T., Kjerstad A., Guttvik A., Taksdal T. & Falk K. (2004a) Heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L.: a new infectious disease. Journal of Fish Diseases 27, 351-358.
Eliassen T. M., Solbakk I. T., Evensen Ø. & Gravningen K. (2004) Isolation of heart and poster skeletal muscle inflammation virus (HSMIV) from salmon. 6th International Symposium on Viruses of Lower Vertebrates, Hokkaido, Japan.
Watanabe K., Karlsen M., Devold M., Isdal E., Litlabo A. & Nylund A. (2006) Virus-like particles associated with heart and skeletal muscle inflammation (HSMI). Diseases of Aquatic Organisms 70, 183-192.
Kongtorp R. T., Halse M., Taksdal T. & Falk K. (2006) Longitudinal study of a natural outbreak of heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L. Journal of Fish Diseases 29, 233-244.
Studies with experimental transmission of heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L. Kongtorp R T, Taksdal T. J Fish Dis. 2009 March; 32(3):253-62. Epub 2009 Feb. 18. PMID: 19236557

## Claims

1. A Piscine Reovirus (PRV) immunogenic composition comprising a PRV cDNA comprising the sequence of any of SEQ ID NOs: 1-10.

2. The immunogenic composition of claim 1 further comprising at least one excipient, additive or adjuvant.

3. The immunogenic composition of claim 1 further comprising at least one polypeptide, or fragment thereof, from an additional virus.

4. A method of inducing an immune response in an animal, the method comprising administering the PRV immunogenic composition of claim 1.

5. A method for preventing, or reducing PRV infection in an animal, the method comprising administering to the animal the PRV immunogenic composition of claim 1.

6. The method of claim 4, wherein the administration is oral administration, immersion administration or injection administration.

7. The method of claim 5 wherein the administration is oral administration, immersion administration or injection administration.