# Patent

## Title
Interactions of betanodaviruses in infection

## Abstract

Disclosed are interactions between different betanodaviruses during infection of cells “in vitro” and in European sea bass. More specifically, fish administered striped jack nervous necrosis viruses (SJNNV), which are then exposed to red-spotted grouper nervous necrosis viruses (RGNNV), have fewer symptoms of disease associated with RGNNV and/or increased survival as compared to fish not administered SJNNV prior to exposure to RGNNV.

## Background
The present application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2014/060852, filed on May 26, 2014 and published in English as International Patent. Publication WO2014/191363 A1 on Dec. 4, 2014, which claims benefit of priority to European Patent Application Ser. Nos. 13169590.0, filed May 28, 2013, and 13169591.8, filed May 28, 2013; all of which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention generally relates to Striped jack nervous necrosis virus (SJNNV) for use in protecting fish against disease associated with Red-spotted grouper nervous necrosis virus (RGNNV) infection. In another aspect the invention relates to a method for protecting fish against disease associated with RGNNV infection, comprising administering SJNNV to the fish, where the fish subsequently exposed to the RGNNV have fewer and/or reduced symptoms of disease as compared to fish which are exposed to RGNNV in absence of prior administration of SJNNV.
BACKGROUND OF THE INVENTION
Viruses of the family Nodaviridae, genus Betanodavirus (i.e., betanodaviruses) infect many different marine and freshwater fish species worldwide and are the etiological agents of viral encephalopathy and retinopathy (VER) in fish. This disease, also called viral nervous necrosis (VNN), can cause significant fish mortality.
The betanodaviruses that cause the disease are non-enveloped and their genomes contain two single-stranded, positive-sense RNA molecules, designated RNA1 and RNA2. Infected cells contain three ssRNAs: RNA1, RNA2 and subgenomic RNA3 derived from RNA1. The T4 variable region within the RNA2 segment has been used to classify betanodaviruses into four different genotypes: striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus (TPNNV), barfin flounder nervous necrosis virus and red-spotted grouper nervous necrosis virus (RGNNV) (Nishizawa et al. 1997). The NNV part of the name refers to nervous necrosis virus and the initial part of the name refers to the fish from which it was isolated. These have become accepted species, with the Striped jack species being known as the type species, based on the characterisation of the SJNag93 isolate. (Iwamoto et al, 2001) Others which may be members of the genus Betanodavirus, but have not yet been approved as separate species, and may or may not be variants of any of the four accepted species listed above include: Atlantic cod nervous necrosis virus, Atlantic halibut nodavirus, Dicentrarchus labrax encephalitis virus, Dragon grouper nervous necrosis virus, Greasy grouper nervous necrosis virus, Japanese flounder nervous necrosis virus, Lates calcarifer encephalitis virus, Malabaricus grouper nevous necrosis virus, Seabass nervous necrosis virus, Solea senegalensis nervous necrosis virus and Turbot nodavirus. (Virus Taxonomy, 2012).
Different genotypes of betanodaviruses likely infect different host ranges of fish. However, coexistence of SJNNV and RGNNV, within single fish in the wild, has been shown in wild meager (Argyrosomus regius). Genetic analysis based on both RNA1 and RNA2 has also demonstrated the existence of reassortant viruses (RGNNV/SJNNV and SJNNV/RGNNV) in infected fish. Such reassortant virus strains or isolates, containing one genome RNA molecule from SJNNV and one genome RNA molecule from RGNNV, have been isolated from Senegalese sole and gilt-head sea bream. Reassortant strains have been associated with clinical outbreaks of disease. Reassortants could result from coinfection of the same cells with SJNNV and RGNNV. Although this evidence indicates that both SJNNV and RGNNV RNA can coexist in the same infected cells, nothing is known about how this affects replication and/or progeny virus production, or how it affects the ability to cause symptoms of disease.
SJNNV and RGNNV have separately been detected in fish species inhabiting the Iberian Peninsula, such as European sea bass (Dicentrarchus labrax), Senegalese sole (Solea senegalensis), gilt-head seabream (Sparus aurata), red-banded seabream (Pagrus auriga), common seabream (Pagrus pagrus), Shi drum, (Umbrina cirrosa) and white seabream (Diploidus sargus).
The RGNNV genotype appears to be the most common in the Mediterranean region and has been responsible for devastating losses in the fishing industry, especially in Greece. The losses in the field of European sea bass have been reported to be up to 60%. Hence there is an urgent need for ways to protect fish, especially sea bream, from the effects of RGNNV infection.
SUMMARY
In a first aspect, the invention relates to Striped jack nervous necrosis virus (SJNNV) for use in protecting fish against disease associated with Red-spotted grouper nervous necrosis virus (RGNNV) infection.
In a preferred embodiment the SJNNV is

    
    
        i) SJNNV with a nucleotide or amino acid sequence which is preferably at least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with the nucleotide or amino acid sequence of SJNNV isolate SJNag93,
        or ii) SJNNV which serologically reacts with antiserum to isolate SJNag93
        or iii) SJNNV with a nucleotide or amino acid sequence which is preferably at least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with the nucleotide or amino acid sequence of SJNNV isolate SJNag93 and serologically reacts with antiserum to isolate SJNag93.
    
    


In a further preferred embodiment the percentage identity of the nucleotide or amino acid sequence of SJNNV is relative to the T4 variable region within the RNA2 segment of SJNNV isolate SJNag93.
In a more preferred embodiment the percentage identity of the nucleotide sequence of SJNNV is relative to the sequence from nucleotides 604 to 1030 of RNA2 of SJNag93 SJNNV, or the percentage identity of the amino acid sequence of SJNNV is relative to amino acids 204 to 331 of the protein encoded by RNA2 of SJNag93 SJNNV, preferably from amino acids 223 to 331 of the protein encoded by RNA2 of SJNag93 SJNNV, more preferably from amino acids 235 to 315 of the protein encoded by RNA2 of SJNag93 SJNNV.
Preferably the SJNNV is selected from the group consisting of SJNag93, Jp/06/SJ, SJOri, SJ91 Nag, SJ92Nag, SJ94Nag and RS95Hir 3, preferably SJNag93.
In a preferred embodiment the SJNNV is administered to the fish in an amount of at least about 1×104 TCID50/fish.
Also preferably, protecting fish against disease involves reducing the mortality in a fish population that results from a RGNNV infection.
Furthermore preferably the fish is selected from the group consisting of European sea bass, Senegalese sole, gilt-head sea bream, red-banded seabream, common seabream, white seabream and wild meager, preferably European sea bass.
In a further preferred embodiment, the RGNNV is
i) RGNNV with a nucleotide or amino acid sequence which is preferably at least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with RGNNV isolate ERV378/102-5/04,
or ii) RGNNV which serologically reacts with antiserum to isolate ERV378/102-5/04,
or iii) RGNNV with a nucleotide or amino acid sequence which is preferably least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with the nucleotide or amino acid sequence of RGNNV isolate ERV378/102-5/04 and serologically reacts with antiserum to isolate ERV378/102-5/04.
In a preferred embodiment the percentage identity of the nucleotide or amino acid sequence of RGNNV is relative to the T4 variable region within the RNA2 segment of RGNNV isolate ERV378/102-5/04.
In yet another preferred embodiment the invention relates to SJNNV for use comprising administering SJNNV to the fish, where the fish subsequently exposed to the RGNNV have fewer and/or reduced symptoms of disease as compared to fish which are exposed to RGNNV in absence of prior administration of SJNNV.
In another aspect the invention relates to SJNNV for use against viral nervous necrosis or viral encephalopathy and retinopathy in fish caused by RGNNV infection.
In yet another aspect the invention relates to a method for protecting fish against disease associated with RGNNV infection, comprising administering SJNNV to the fish, where the fish subsequently exposed to the RGNNV have fewer and/or reduced symptoms of disease as compared to fish which are exposed to RGNNV in absence of prior administering of SJNNV.
In a preferred embodiment the SJNNV is used to decrease mortality of fish, preferably sea bass, which are later exposed to RGNNV, compared to fish, preferably sea bass, that have not been administered with the SJNNV or a virus which is preferably at least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with SJNNV isolate SJNag93.
In another preferred aspect of the invention there is provided method for reducing mortality in fish, preferably sea bass, that is associated with RGNNV infection, comprising inoculating the fish, preferably sea bass, with SJNNV, wherein the fish, preferably sea bass, which are subsequently exposed to a strain or strains of RGNNV, that is capable of causing mortality, have a reduced mortality rate as compared to sea bass that have not been inoculated with the SJNNV and are exposed to that strain or strains of RGNNV.
Preferably in the method according to the invention the RGNNV is capable of causing mortality in the fish. Preferably also the fish administered the SJNNV and subsequently exposed to the RGNNV have lower mortality as compared to fish exposed to RGNNV in absence of prior administering of SJNNV.
In a preferred embodiment of the method of the invention the fish are exposed to the RGNNV up to about 6 weeks after administering the SJNNV. More preferably the fish are exposed to the RGNNV up to about 3 weeks after administering the SJNNV. Especially preferably the fish are exposed to the RGNNV up to about 72 hours after administering the SJNNV. More especially preferably the fish are exposed to the RGNNV up to about 24 hours after administering the SJNNV. In another preferred embodiment the SJNNV includes isolate SJ93Nag. In yet another preferred embodiment where the SJNNV and the RGNNV are capable of infecting the same species of fish, which preferably include European sea bass, Senegalese sole, gilt-head sea bream, red-banded seabream, common seabream, white seabream and wild meager. In another preferred embodiment of the method of the invention administering of the SJNNV is performed by intramuscular or peritoneal injection of the fish with the virus or immersion of fish in a bath containing the virus. In yet another preferred embodiment of the method of the invention an amount of the SJNNV administered to the fish is at least about 1×104 TCID50/fish. More preferably the amount of the SJNNV administered to the fish is between about 1×104 and about 1.5×104 TCID50/fish. In a preferred embodiment of the method of the invention administering of the SJNNV results in increased transcriptional expression of at least one interferon-inducible gene, where preferably the interferon-inducible gene includes Mx. Preferably in the method according to the invention the sea bass that are inoculated with SJNNV and subsequently exposed to RGNNV have a reduced mortality rate for up to about 72 hours after inoculation with SJNNV.
In a preferred embodiment of the method according to the invention the SJNNV is

    
    
        i) SJNNV with a nucleotide or amino acid sequence which is preferably at least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with the nucleotide or amino acid sequence of SJNNV isolate SJNag93,
        or ii) SJNNV which serologically reacts with antiserum to isolate SJNag93
        or iii) SJNNV with a nucleotide or amino acid sequence which is preferably least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with the nucleotide or amino acid sequence of SJNNV isolate SJNag93 SJNNV and serologically reacts with antiserum to isolate SJNag93.
    
    


In a preferred embodiment of the method according to the invention the percentage identity of the nucleotide or amino acid sequence is relative to the T4 variable region within the RNA2 segment of SJNNV isolate SJNag93.
In a further preferred embodiment of the method according to the invention the RGNNV has a nucleotide or amino acid sequence which is preferably at least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with RGNNV isolate ERV378/102-5/04

    
    
        or ii) RGNNV which serologically reacts with antiserum to isolate ERV378/102-5/04
        or iii) RGNNV with a nucleotide or amino acid sequence which is preferably least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with the nucleotide or amino acid sequence of RGNNV isolate ERV378/102-5/04 and serologically reacts with antiserum to isolate ERV378/102-5/04.
    
    


In a preferred embodiment of the method according to the invention the percentage identity of the nucleotide or amino acid sequence is relative to the T4 variable region within the RNA2 segment of RGNNV isolate ERV378/102-5/04.
In yet another preferred embodiment of the invention the RGNNV has a nucleotide sequence which is preferably at least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with RGNNV RNA1 and RNA2. A representative example of RGNNV RNA1 is to be found in Genbank under the accession number NC_008040.1. A representative example of RGNNV RNA2 is to be found in Genbank under the accession number NC_008041.1.
In a preferred embodiment the percentage identity of the nucleotide sequence is relative to the T4 variable region within the RNA2 segment of RGNNV. A representative example of RGNNV RNA2 is to be found in Genbank under the accession number NC_008041.1.
In another aspect the invention relates to a vaccine comprising SJNNV for use in preventing or treating RGNNV infection in fish. In yet another aspect the invention relates to a vaccine for use in protecting fish against viral nervous necrosis or viral encephalopathy and retinopathy in fish caused by RGNNV infection, wherein the vaccine comprises SJNNV.
Preferably in said vaccine the SJNNV is
i) SJNNV with a nucleotide or amino acid sequence which is preferably at least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with the nucleotide or amino acid sequence of SJNNV isolate SJNag93,
or ii) SJNNV which serologically reacts with antiserum to isolate SJNag93
or iii) SJNNV with a nucleotide or amino acid sequence which is preferably least 90% identical, more preferably at least 95% identical, particularly preferably at least 97% identical, more particularly preferably at least 99% identical with the nucleotide or amino acid sequence of SJNNV isolate SJNag93 and serologically reacts with antiserum to isolate SJNag93. More preferably the percentage identity of the nucleotide or amino acid sequence of the SJNNV is relative to the T4 variable region within the RNA2 segment of SJNNV isolate SJNag93. In a preferred embodiment of the vaccine the SJNNV is administered to the fish in an amount of at least about 1×104 TCID50/fish. In another preferred embodiment of the vaccine SJNNV RNA2 is administered to the fish. Preferably also the SJNNV is composed of SJNNV RNA1 and SJNNV RNA2. In an alternate embodiment, the SJNNV is SJNNV RNA2, without SJNNV RNA1. In a preferred embodiment of the vaccine the SJNNV is in the form of a heat killed or chemically inactivated SJNNV. Typically the chemically inactivated SJNNV is inactivated using an aziridine compound, preferably binary ethyleneimine. Preferably the SJNNV is administered in the form of a DNA or RNA vaccine, preferably RNA vaccine. In a another preferred embodiment of the vaccine according to the invention, the fish is selected from the group consisting of European sea bass, Senegalese sole, gilt-head sea bream, red-banded seabream, common seabream, white seabream and wild meager, preferably European sea bass. Preferably also in the vaccine according to the invention the SJNNV is selected from the group consisting of SJNag93, Jp/06/SJ, SJOri, SJ91Nag, SJ92Nag, SJ94Nag and RS95Hir 3, preferably SJNag93.
In another aspect the invention relates to the use of SJNNV in the manufacture of a composition for reducing the amount of mortality in fish that results from infection with RGNNV.
In yet another aspect the invention relates to a method for inhibiting replication of SJNNV in cells, comprising infecting cells with RGNNV prior to, or simultaneously with, infecting the cells with SJNNV.
A further aspect of the invention relates to a method for infecting fish with viruses, comprising:
a) administering SJNNV to the fish; and
b) exposing the fish to RGNNV;
wherein the fish have fewer and/or reduced symptoms that are associated with RGNNV infection than do fish exposed to RGNNV that have not been administered SJNNV.
In yet a further aspect the invention relates to a method for stimulating replication of RGNNV in cells, comprising infecting cells with SJNNV prior to, or simultaneously with, infecting the cells with RGNNV.



BRIEF DESCRIPTION OF THE DRAWINGS
In the accompanying drawings, which are incorporated in and constitute a part of the specification, embodiments of, or related to, methods and compositions are illustrated, which, together with the detailed description given below, serve to describe the examples. It should be appreciated that the embodiments illustrated in the drawings are shown for the purpose of illustration and not for limitation. It should be appreciated that changes, modifications and deviations from the embodiments illustrated in the drawings may be made without departing from the spirit and scope of the invention, as disclosed below.
FIG. 1 illustrates example data from a study where different betanodavirus genotypes were administered to 5 g juvenile sea bass and fish mortality was followed.
FIG. 2 illustrates example data from a study where different strains and inoculums of RGNNV were administered to sea bass and fish mortality was followed. Positive control is RGNNV isolate ERV378/102-5/04.
FIG. 3 illustrates example data from a study where an RGNNV strain was administered to sea bass of different weights and fish mortality was followed.
FIG. 4 illustrates example data from a study where an RGNNV strain was administered to sea bass that had already been administered SJNNV (e.g., RGNNV superinfection of fish infected with SJNNV). Fish mortality was followed.
FIG. 5 illustrates example data from a study where RGNNV genome RNA was quantified at various times after fish infected with SJNNV had been superinfected with RGNNV. First column at each time point is the RG control group (group 3) and second column at each time point is the SJ/RG group (group 4).
FIG. 6 illustrates example data from a study where SJNNV genome RNA was quantified at various times after fish infected with SJNNV had been superinfected with RGNNV. First column at each time point is the SJ control group (group 2) and second column at each time point is the SJ/RG group (group 4).
FIG. 7 illustrates example data from studies where SJNNV genome RNA was quantified at various times after: (A) cells were coinfected with SJNNV and RGNNV, (B) cells infected with SJNNV were superinfected with RGNNV, and (C) cells infected with RGNNV were superinfected with SJNNV.
FIG. 8 illustrates example data from studies where RGNNV genome RNA was quantified at various times after: (A) cells were coinfected with SJNNV and RGNNV, (B) cells infected with SJNNV were superinfected with RGNNV, and (C) cells infected with RGNNV were superinfected with SJNNV.
FIG. 9 SJNNV RNA 1 (SEQ ID No. 1) Striped Jack nervous necrosis virus gene for protein A and protein B, complete cds, nucleotide sequence
FIG. 10a SJNNV RNA 1 (SEQ ID No. 2) Amino acid sequence of protein A
FIG. 10b SJNNV RNA 1 (SEQ ID No. 3) Amino acid sequence of protein B
FIG. 11 SJNNV RNA 2 (SEQ ID No 4) Striped Jack nervous necrosis virus gene for coat protein, complete cds, nucleotide sequence
FIG. 12 SJNNV RNA 2 (SEQ ID No 5). Amino acid sequence of coat protein



DETAILED DESCRIPTION
Herein, “SJNNV” means a betanodavirus of the striped jack nervous necrosis virus species or genotype. The SJNNV viruses disclosed herein are generally able to infect, or at least adsorb to, at least some of the same cells as some viruses of the RGNNV species or genotype. Example of SJNNV isolates include, but are not limited to, SJ93Nag, Jp/06/SJ, SJOri, SJ91Nag, SJ92Nag, SJ94Nag and RS95Hir. A preferred example of an SJNNV is isolate SJ93Nag. (See Genbank sequence AB056571, FIG. 9: SEQ ID No. 1 (RNA 1: virus genes for protein A, protein B), FIG. 10a: SEQ ID NO. 2: Amino acid sequence of protein A [Striped Jack nervous necrosis virus] GenBank: BAB64329, FIG. 10b: SEQ ID NO. 3: Amino acid sequence of protein B [Striped Jack nervous necrosis virus] GenBank: BAB64330. FIG. 11 SEQ ID No 4 Striped Jack nervous necrosis virus gene for coat protein, complete cds GenBank: AB056572. FIG. 12 SEQ ID No 5. Amino acid sequence of Striped Jack nervous necrosis virus gene for coat protein, complete cds GenBank: BAB64331)
Herein, “RGNNV” means a betanodavirus of the red-spotted grouper nervous necrosis virus species or genotype. The RGNNV viruses disclosed herein are generally able to infect, or at least adsorb to, at least some of the same cells as some viruses of the SJNNV genotype. Examples of RGNNV isolates include, but are not limited to ERV378/102-5/04, SpDI-IAusc1688.08, Mt/01/Sba, Gr/02/Sba, Gr/12/Sba, Pt/08/Sba, It/23/Sba, It/24/Sdr, It/19/Sba, Jp/15/Rp, Th/07/Bgr, Sg/14/Bar, Sp/20/Sba, Gr/16/Sba, SGWak97, SGMie95, RGOka94, JSOit98, KGOit97, HG0001, BGThA99, SBGre96, WSBUS99A, WSBUS99B, BAus94, JFHir92, JFHir96, JF93Hir, MR94Tha, RG94Oka, JF94Wak, JF95Oit, RG91Tok, SB95Ita, SG94Oit, JF95Tok, JS95Shi, JF95Sag, PA94Oit and KG95OIt. A preferred example of an RGNNV is isolate is ERV378/102-5/04 or SpDI-IAusc1688.08, particularly preferably ERV378/102-5/04.
Herein, “fish,” generally refers to species of fish that can be infected by SJNNV and RGNNV. These species may include, but may not be limited to, European sea bass, Senegalese sole, gilt-head seabream, red-banded seabream, common seabream, white seabream Shi drum, and wild meager.
Herein, “capable of causing,” generally as referred to viruses, means that a virus can produce the specified effect (e.g., symptoms and/or disease, and/or mortality) under at least certain conditions or circumstances.
Herein, “symptoms,” refers to observable and/or measurable characteristics of or in an animal that are generally thought not to be normal for a healthy animal. Symptoms may be indicative of disease. Herein, the animal will generally be a fish and symptoms may be changes in characteristics of fish (e.g., physical changes, behavioral changes, etc.) that are different than what are thought to be normal or healthy characteristics. Herein, symptoms may be attributed to or considered to be caused by infection with a virus. For example, betanodaviruses can be the cause of viral nervous necrosis (VNN) in fish. Symptoms of VNN may include vacuolation in cells of the retina and central nervous system neuronal degeneration in the central nervous system (CNS), anorexia, abnormalities in pigmentation, sight, bladder control and swim behavior. VNN caused by betanodaviruses may result in death of infected fish. Mortality, therefore, could be considered to be a symptom of VNN, although mortality may be referred to as a result of VNN. Symptoms may be referred to using other terms, such as clinical signs, manifestations of disease, and the like.
Herein, “reducing symptoms,” “reducing mortality,” “reducing mortality rate,” and the like, generally refer to comparison of properties in different fish, or different groups of fish, where the extent of the observed properties in one group of fish is decreased or reduced in another group. Generally, the decrease can be attributed to an action or characteristic. Herein, for example, a population of fish infected with RGNNV generally will display symptoms due to infection with RGNNV. A measurable number of the infected fish may die. The number of fish in a comparable population that display the symptoms or die, where the fish have been administered SJNNV prior to infection with RGNNV, may be less than in the group that was not administered SJNNV. In this situation, prior administration of SJNNV may be said to have reduced the symptoms and/or mortality caused by or associated with RGNNV. Reductions in properties like symptoms and mortality, due to SJNNV administration, may indicate protection against disease associated with or caused by RGNNV.
Herein, “administering” or “inoculating,” generally as referred to viruses and their interaction with fish and/or cells, means that virus is intentionally given to, applied to or introduced into, the fish or cells, such that the viruses can infect, or at least adsorb to, cells in the fish or cells cultured in vitro. One method of administering or inoculating virus is by injection of virus into the body of a fish.
Herein, “exposure to” or “exposed to,” generally refers to a situation where an animal (e.g., a fish) is in proximity to an infectious agent (e.g., a virus) such that there is a possibility that or opportunity for the virus to infect cells of the animal.
Herein, “subsequently administering” or “subsequently inoculating,” generally refers to a situation where a virus is administered to a fish at a time after some other event has occurred. In one example, one virus (e.g., RGNNV) is administered to a fish at a point in time after a different virus (e.g., SJNNV) has been administered to the fish. In this situation, RGNNV may be said to have been “subsequently administered” in relation to the earlier administration of SJNNV.
Herein, “infection,” refers to the process whereby a virus interacts with a host cell and is multiplied, or at least has the opportunity to multiply, within the host cell such that additional viruses are produced. It is possible for the infection process to be interrupted or blocked at various stages. When the infection process is interrupted or blocked, production of additional viruses may be inhibited or reduced.
Herein, “coinfection,” generally refers to the process whereby different viruses (e.g., a SJNNV and a RGNNV) interact with the same host cell at approximately the same time or simultaneously. In one example, coinfection of a cell by two different viruses results in coexistence of genomes from both viruses inside of a host cell.
Herein, “superinfection,” refers to the situation where two viruses interact with the same host cell at approximately different times. For example, in the instance where cells are exposed to SJNNV such that an infection of cells with SJNNV may be initiated and, at some later time, the cells are exposed to RGNNV such that an infection with RGNNV may be initiated, SJNNV may be said to be the “infecting” virus and RGNNV may be said to be the “superinfecting” virus. Superinfection, therefore, generally refers to infection of cells with a virus where the cells have previously been infected with a virus. An infecting virus may be said to have infected cells prior to a superinfecting virus. A superinfecting virus may be said to have infected cells subsequent to an infecting virus.
Herein, “replication,” generally refers to the process where viral genomes are reproduced in the interior of a host cell. Generally, replication includes a subset of the events that occur during infection.
Herein, “reassortant,” generally refers to a virus with a multipartite genome (e.g., a genome in multiple segments) where the origin of at least two genome segments can be attributed to viruses that are not the same. For example, betanodaviruses are known that appear to have one genome RNA molecule that originated from SJNNV and a second genome RNA molecule that originated from RGNNV.
Herein, “transcriptional expression,” refers to synthesis of RNA from a DNA copy of a gene. In one example, steady-state levels of RNA encoded by specific genes within cells may be measured and used to infer regulation of expression of the gene at the level of transcription. In one example, levels of RNA encoded by a gene is measured in different populations of cells, and differences in the RNA levels is attributed to a change in the rate of transcription of that gene.
Disclosed herein are interactions between, and the effects of interactions between, betanodaviruses in infection of fish, or cells in vitro. The betanodaviruses described are generally viruses of the SJNNV species and viruses of the RGNNV species. In one example, infection of fish with an SJNNV can reduce or diminish disease, symptoms of disease, effects of disease, and the like, of a superinfecting RGNNV, as compared to disease/symptoms/effects of an RGNNV in absence of a prior SJNNV infection.
The effect of infecting SJNNV on superinfecting RGNNV may be seen in a variety of species of fish. Generally, the fish include any species that can be infected by SJNNV and RGNNV. Some examples include European sea bass (Dicentrarchus labrax), Senegalese sole (Solea senegalensis), gilt-head sea bream (Sparus aurata), red-banded seabream (Pagrus auriga), common seabream (Pagrus pagrus) and white seabream (Diploidus sargus).
Generally, the SJNNV may be an SJNNV that can infect a fish species that RGNNV can also infect. In one example, the infecting SJNNV may cause fewer or less severe symptoms than does a superinfecting RGNNV. In one example, SJNNV may be the SJ93Nag strain or isolate (Iwamoto et al, 2001). Other SJNNV isolates may include SJOri, SJ91Nag, SJ92Nag, SJ94Nag and RS95Hir.
“Serologically reacts with antiserum” may be tested by methods well known in the art. Examples of tests for detecting serological cross reaction are ELISA, hemagglutination inhibition (HI), serum neutralization (SN) assay, cross neutralization test and virus neutralization test. A preferred example is the cross neutralization test. See Mori et al., 2003, p21 for the methodology used to test serological cross reaction with antiserum for nodavirus isolates.
SJNNV may be purified from diseased larvae of Striped jack fish and the RNA extracted by methods well known in the art, for example as described by Nishizawa et al 1995, p1564.
Identity of the RNA virus as belonging within the SJNNV genotype according to the invention may be established by comparison with the variable region of the SJNag93 coat protein gene sequence from nucleotides 604 to 1030 of RNA2 (SEQ ID NO: 4), or at the amino acid level aa 204 to 331 of SEQ ID NO: 5, preferably 223 to 331, more preferably 235 to 315, as described by Nishizawa et al., 1997, particularly on page 1635, and Nishizawa et al., 1995 and Skiliris et al., page 64.
Infectious RNA transcripts for SJNNV may alternatively be made by methods well known in the art, based on the known sequences of SJNNV (See seq ID No. 1-5). This has been descried in detail in Iwamoto et al 2001, p2654-2656.
Generally, the RGNNV may infect a fish species that SJNNV can also infect. In one example, RGNNV causes symptoms and/or disease in the infected fish. In one example, RGNNV can cause mortality in the infected fish. An example RGNNV strain or isolate is ERV378/102-5/04 (Lopez-Jimena et al., 2011).
The betanodaviruses may be propagated by a variety of methods. For example, the viruses may be grown in cultured cells. A variety of cultured cells may be used. Some example cell lines include the E-11 cell line (ECACC, No. 01110916; Iwamoto et al., 2000), SAF-1 cells (ECACC, No. 00122301; Aquaculture 150:143-153, 1997) and SSN-1 cells (J. Gen. Virol. 77, 2067-2071, 1996). Techniques for propagating the viruses are well known in the field.
Administration of viruses, including SJNNV, to fish may be performed using a variety of techniques. For example, SJNNV may be administered to larvae and/or juveniles of a fish species. SJNNV may be administered to adult or mature fish. In one example, fish are between about 2.5 g and 10 g in weight. Routes of administration of SJNNV may vary. Examples include intramuscular injection, intraperitoneal injection, orally in feed, immersion of fish in a bath containing the virus, and others.
Generally, the amount of SJNNV administered to fish is an amount that reduces symptoms, including mortality, that may be caused by RGNNV. The “amount” of virus may be determined by a variety of techniques. Generally, assays that measure infectious activity of viruses may be used. In one example, infectious virus may be measured by determining TCID50. Other methods may measure plaque-forming units. Additional methods may be used. The amount of SJNNV administered may be 102, 103, 105, 106, 107, or more TCID50/fish. In one example, the amount of SJNNV administered to fish is at least about 1×104 TCID50/fish. In one example, the amount of SJNNV administered to fish is between about 1×104 and 1.5×104 TCID50/fish. Example volumes infected into the fish may be 10, 20, 50, 100, 150, 200, 250 or 500 μl. In one example, the volume is between about 10 and 200 μl. In another example, 50 or 100 μl is injected.
Formulation of the viruses for administration to fish may be performed using a variety of methods. Generally, the formulation will preserve the infectivity of the viruses until the time the viruses are administered to fish. In one example, viruses may be formulated in a biological buffer.
The effect SJNNV has on reducing symptoms in fish that are caused by RGNNV generally may be long lasting. For example, fish administered SJNNV may display reduced symptoms due to subsequent RGNNV infection for various times after administering SJNNV. In various examples, fish administered SJNNV may have reduced symptoms due to subsequent RGNNV infection, as compared to fish not administered SJNNV, for 6, 5, 4, 3, 2 or 1 week(s) after administration of SJNNV. The reduced symptoms may have durations of 6, 5, 4, 3, 2 or 1 day(s) after administration after SJNNV. The reduced symptoms may have durations of 96, 72, 48, 24, 12 or 6 hours after administration of SJNNV.
No mechanism of action for the effect of SJNNV on disease and/or mortality associated with RGNNV is relied upon or offered. It is merely disclosed that example results indicate that infection of fish and/or cells of or from fish may result in or be associated with changes in expression of one or more genes known to be regulated by interferon. In one example, infection of fish with SJNNV is associated with increased levels of steady-state RNA from one or more interferon-inducible gene. In one example, the interferon-inducible gene may be Mx. In one example, SJNNV is associated with increased transcription from the interferon-inducible genes.
In one example, infection of cells with RGNNV prior to, or simultaneously with, SJVVN may result in inhibition of replication of SJNNV in the cells. SJNNV superinfection of cells infected with RGNNV may cause inhibition of genome replication of SJNNV. RGNNV and SJNNV coinfection of cells may cause inhibition of genome replication of SJNNV.
In one example, infection of cells with SJNNV prior to, or simultaneously with, RGNNV may result in stimulation of replication of RGNNV in cells. RGNNV superinfection of cells infected with SJNNV may cause stimulation of genome replication of RGNNV. SJNNV and RGNNV coinfection of cells may cause stimulation of genome replication of RGNNV.
EXAMPLES
The examples are for the purpose of illustrating an example and are not to be construed as illustrating limitations.
Example 1—Betanodaviruses, Cell Line and Virus Growth
Viral isolates ERV378/102-5/04 (RGNNV genotype; Lopez-Jimena et al, 2011), SpDI-IAusc1688.08 (RGNNV genotype; Olveira et al, 2009), SJ93Nag (SJNNV genotype, Iwamoto et al, 2001), and SpSs-IAusc160.03 (reassortant with RGNNV-type RNA1 and SJNNV-type RNA2; Olveira et al, 2009) were grown at 25° C. on the E-11 cell line (European Collection of Cell Cultures, UK, Cat. No. 01110916) (Iwamoto et al., 2000) according to Lopez-Jimena et al., 2011. Cells were grown at 25° C. in Leibovitz (L-15) medium (Invitrogen) supplemented with 1% penicillin-streptomycin (Invitrogen) and 5% fetal bovine serum, until semi-confluent, before virus inoculation. Virus titers were determined by using the highest dilution causing cytopathic effect in 50% of the inoculated cultures (TCID50) as described (Tissue Culture: Methods and Applications; Kruse and Patterson, eds.; pp. 527-532, Academic Press, New York, 1973).
Example 2—Sea Bass
European sea bass (Dicentrarchus labrax) were maintained at between 21-25° C. Sea bass of the indicated weights were inoculated with viruses by intramuscular (i.m.) injection (0.1 ml of L-15 medium was used as control inoculum for controls). In other studies, sea bass were inoculated by immersion of fish in a bath that contained virus (L-15 medium used as inoculum for controls).
Example 3—Sea Bass Mortality Caused by Some RGNNV Strains
European sea bass of 2.5 g average weight were injected i.m. with 1.0×106, 2.5×105 1.0×105 or 1.0×104 TCID50/fish of the viral isolate SpDI-IAusc1688.08 (RGNNV genotype). Fish were similarly injected with 2.5×105 TCID50/fish of the viral isolate ERV378/102-5/04 RGNNV genotype (listed as positive control in FIG. 2). The ERV378/102-5/04 isolate caused a cumulative mortality rate of 69% in this study.
In another experiment (Lopez-Jimena et al., 2011) it was shown that, in juvenile European sea bass (mean weight 10±0.3 g; n=100) that were challenged with 106 TCID50 of the ERV378/102-5/04 RGNNV isolate by i.m. injection, cumulative fish mortality was 37%. Analyses of tissue homogenates (brain and eyes) from dead fish by cell culture inoculation demonstrated the presence of RGNNV in all the samples analyzed. Identity of the virus was subsequently confirmed using RT-PCR.
Example 4—Different Betanodavirus Genotypes and Fish Mortality
Sea bass 5 g weight were i.m. challenged with the following VNN isolates: SJ93Nag (SJNNV genotype), isolate ERV378/102-5/04 (RGNNV), and SpSs-IAusc160.03 (reassortant RGNNV RNA1 and SJNNV RNA2). A volume of 0.1 ml L-15 medium was injected in the control group, whereas the dose of 2.5×105 TCID50/fish of the different virus was used. Temperature was measured daily and maintained between 22-25° C. approximately. Typical symptoms of VNN and mortality were only recorded in fish inoculated with the RGNNV (47% cumulative mortality), and reassortant (33% cumulative mortality). No mortality was recorded from the SJ93Nag or the control group (FIG. 1).
Example 5—Determination of the Anti-VNNV Antibodies in Fish Survivors
Sera from 5 fish survivors from Example 4 were pooled and three pools were screened in order to detect specific anti-betanodavirus antibodies by ELISA. ELISA plates were coated with either SJ93Nag (SJNNV genotype), SpSs-IAusc160.03 (reassortant RGNNV RNA1/SJNNV RNA2) or RGNNV (Lopez-Jimena et al., 2011) and pooled sera (1/32 dilution) were analysed in triplicate. The optical density (OD) was determined at 450 nm. The OD mean of the negative control wells (PBS instead of fish serum) (three times repetitive) was the cut-off threshold considered.
Table 1 shows the mean OD values and standard deviation (SD) of the three replicates. The cut-off threshold value was 0.305.







TABLE 1





Mean OD values and standard deviation (SD) of specific


anti-VNN antibody detection using ELISA





















Sera 2 g
Sera 2 g




Sera 2 g
seabass
seabass
Sera 2 g



seabass
CONTROL
CONTROL
seabass



CONTROL
30 dpi
0 dpi
ANTIGEN





MEAN
0.026472222
0.041805556
0.037916667
SJ93Nag


SD
0.005738112
0.009912581
0.013670731


MEAN
0.026583333
0.040805556
0.04625
RG RNA1/






SJ RNA2


SD
0.006437736
0.008921468
0.004714045


MEAN
0.039361111
0.052694444
0.061583333
RGNNV


SD
0.005853141
0.010286633
0.017913372







Sera 5 g



Sera 5 g
seabass
Sera 5 g



seabass
RG RNA1/
seabass



SJ93Nag
SJ RNA2
RGNNV
ANTIGEN





MEAN
0.83281944
0.871597222
0.83670833
SJ93Nag


SD
0.07542865
0.085379111
0.07819065


MEAN
0.82926389
0.86881944
0.84126389
RG RNA1/






SJ RNA2


SD
0.10151373
0.067798995
0.11232756


MEAN
0.05815278
0.126152778
0.42470833
RGNNV


SD
0.02060295
0.079490973
0.09031488









High levels of viral genome and infective viral particles were recorded in the brain of survivor fish inoculated with the SJNNV isolate, although there was no mortality or clinical signs. Specific antibody response, measured by indirect ELISA, was only observed in the VNN-innoculated groups, with titres of 1/1024, 1/4096 and 1/8192 for RGNNV, SJNNV and the reassortant inoculated fish respectively.

Example 6—Effect of Sea Bass Size on Mortality
European sea bass of 2.5 g average weight were injected i.m. with 2.5×105 TCID50/fish of the viral isolate ERV378/102-5/04 (RGNNV genotype). Fish mortality was recorded daily. The ERV378/102-5/04 isolate of RGNNV caused a cumulative mortality rate of 69%. As mentioned in Example 4, the same inoculation to sea bass of average weight 5 g resulted in 47% cumulative mortality. The data for the 2.5 and 5 g fish are shown in FIG. 3. In third trial, inoculation of 106 TCID50 of the same virus to sea bass of average weight 10 g resulted in 37% mortality. Fish size is therefore inversely related with mortality rate for infection with RGNNV genotype.
Example 7—Superinfection Experiments in Fish
Studies were performed to monitor the course of disease in sea bass infected with RGNNV, when the sea bass had previously been infected with SJNNV.
Juvenile European sea bass (average weight 10-15 g) were distributed into four separate tanks of 600 L:
Non-infected control group (n=150): i.m. injected with L-15 medium (0.1 ml) at time 0 and again 24 hours later;

    
    
        1) Non-infected control group (n=150): i.m. injected with 0.1 ml of L-15 medium at time 0 and 25 hours later;
        2) SJNNV-infected control group (n=150): i.m. injected with 1.5×106 TCID50 SJNNV (isolate SJ93Nag) at time 0 and with L-15 medium (0.1 ml) 24 hours later;
        3) RGNNV-infected control group (n=150): i.m. injected with L-15 medium (0.1 ml) at time 0 and with 1.5×106 TCID50 RGNNV (isolate ERV378/102-5/04) 24 hours later;
        4) Superinfected group (n=150): i.m. injected with 1.5×106 TCID50 SJNNV (isolate SJ93Nag) at time 0 and then with 1.5×106 TCID50 RGNNV (isolate ERV378/102-5/04) 24 hours later.
    
    


From each experimental group, 50 fish were kept separately for use to determine cumulative mortality. The remaining 100 fish were used for sampling (see data in Examples 8-10). Water temperature was maintained between 22 and 24° C. over the course of the trial. Mortality was recorded daily and dead fish were frozen at −80° C. for use in later virological examinations.
For the SJNNV-infected control group (Group 2), as well as in the non-infected control group (Group 1), no mortality was observed (FIG. 4).
For the RGNNV-inoculated control group (Group 3), mortality was first detected at about 6 days post infection. Mortality stabilized at about 76%, 15 days post infection.
In the superinfected group (Group 4), although fish exhibited dark coloration and abnormal swimming behavior, only 2 animals died (4% cumulative mortality). In this group, SJNNV infection dramatically reduced the mortality seen when RGNNV infected fish without prior SNJJV infection (Group 3).
Example 8—Virus From Dead RGNNV-Infected Fish in the Superinfection Experiments
Dead fish from the RGNNV-infected control group (Group 3), from the experiment described in Example 7, were assayed for the presence of virus. Brain and eye tissue from fish found dead on day 6 and on day 12 post infection, were pooled, and homogenized in 20% (w/v) L-15 medium supplemented with 1% penicillin-streptomycin and 2% fetal bovine serum. Homogenates were treated with 10% penicillin-streptomycin at 4° C. overnight. Homogenates were then centrifuged twice at 7,500×g at 4° C. for 15 min, and 100 μl of the supernatants were used for virus titrations on E-11 cells to determine TCID50. The data are shown in Table 2, below.







TABLE 2







Viral titers from dead fish from the RGNNV-infected control group










Days post infection fish found dead
Viral titer (TCID50/g)














6
1.6 × 109



12
1.6 × 108










These data indicate that the virus could be isolated from the dead fish.
Example 9—Quantification of Viral Genomes from SJNNV-Infected, RGNNV-Superinfected Fish in Superinfection Experiments
Quantification of viral genomes in fish from the superinfected group (Group 4), from the experiment described in Example 7, was performed. Nine live fish were randomly collected from Group 4 at 12 hours, 3 days, and 7 days after the RGNNV superinfection. Fish were killed by an overdose of anaesthetic (MS-222, Sigma), and the brain and eyes from the 3 fish at each time point were pooled together, immediately frozen in liquid nitrogen, and stored at −80° C. until used.
The pooled organs were homogenized in L-15 medium (20% w/v) supplemented with 1% penicillin-streptomycin and 2% FBS. Homogenates were centrifugated twice at 7,500×g at 4° C. for 15 min. Volumes of 200 μl of the three clarified homogenates, at every sampling time, were used for total RNA extraction.
RNA extraction was performed using Trizol® (Invitrogen). RNA concentration obtained was determined at 260 nm using the ND-1000 system (NanoDrop Thermo Scientific). RNA was stored at −80° C. until used.
Synthesis of cDNA from the RNA was performed using the Transcriptor First-Strand cDNA Synthesis kit (Roche). The reaction was carried out with 1 μg of total RNA. The cDNA concentration was determined at 260 nm using the ND-1000 system. cDNA was stored at −20° C. until used.
Viral genomes were quantified using SYBR Green I-based absolute real-time PCR (qPCR) protocols detecting, separately, the RNA2 segments of the RGNNV and SJNNV genotypes. Specifically, RGNNV RNA2 was quantified using a procedure already described (Lopez-Jimena et al., 2011). SJNNV RNA2 segment was quantified using the primers SJ-RNA2-F (5′-GACACCACCGCTCCAATTACTAC-3′, nucleotides 665-687) (SEQ ID NO. 6) and SJ-RNA2-R (5′-ACGAAATCCAGTGTAACCGTTGT-3′, nucleotides 739-717) (SEQ ID NO. 7), that amplify a 75-bp fragment within the T4 region (GenBank accession number D30814), using the PCR conditions already described (Lopez-Jimena et al., 2011).
Significant differences between the number of copies of each viral genome segment at different times p.i. were calculated by one-way ANOVA, followed by Fisher's Least Significant Difference (LSD) Test. Statistical analysis was performed using IBM SPSS Statistics software. Values of p<0.05 were considered significant.
For quantification of RGNNV genomes, the data (FIG. 5) showed a significant decrease in RGNNV replication in the Superinfected group (Group 4) at 7 days after superinfection with RGNNV (8 days after infection with SJNNV) as compared with the RGNNV-inoculated control group (Group 3) (FIG. 5).
In contrast, no significant differences were observed when SJNNV replication in the Superinfected group (Group 4) was compared to the SJNNV-infected control group (Group 2) (FIG. 6).
Example 10—Interferon (IFN)-Inducible Gene Expression in Superinfection Experiments
Fish from the experiments described in Example 7 were tested for transcriptional expression of the IFN-inducible gene, Mx, using real time, RT-qPCR. The data showed no transcriptional expression of Mx in fish from the RGNNV-infected control group (Group 3) from 0 to 48 hours post infection. In contrast, Mx transcriptional expression was increased in fish from the SJNNV-infected control group (Group 2) and from the Superinfected group (Group 4). These data indicate that infection of fish by SJNNV may induce the IFN-inducible gene, Mx, while RGNNV does not. These results suggest that the induction of the IFN mediated system by the previous infection with SJNNV could be responsible for the decrease in the mortality recorded in the superinfected group, protecting sea bass from the effects of posterior infection with RGNNV.
Example 11—Superinfection Experiments in Cultured Cells
Studies were performed to investigate the effects of coexistence of SJNNV and RGNNV on the replication and multiplication of each virus. Viral genome copy number in infected cells, and production of infective virus particles produced by infected cells was determined. E-11 cells were grown in 24-wells plates. Second-passage cell monolayers, in duplicate, were infected at a multiplicity of infection of 0.1 at 25° C., a temperature optimum for multiplication of both SJNNV and RGNNV. The experimental groups were as shown in Table 3, below. In each experimental group, non-inoculated E-11 cells were included as a control.







TABLE 3







Experimental groups and sampling times analyzed











Hours postinfection samples


Group
Virus genotype(s) inoculated
taken





1
SJNNV-infected control group
0a, 12, 24, 48, 72, 96


2
RGNNV-infected control group
0a, 12, 24, 48, 72, 96


3
SJNNV + RGNNV
0a, 12, 24, 48, 72, 96



coinfection group


4
SJNNV → 24 h → RGNNV
0a,b, 12b, 24b, 24c, 48c, 72c



superinfection group
(SJNNV)




0a,c, 12c, 24c, 48c, 72c, 96c




(RGNNV)


5
RGNNV → 24 h → SJNNV
0a,d, 12d, 24d, 24e, 48e, 72e



superinfection group
(RGNNV)




0a,e, 12e, 24e, 48e, 72e, 96e




(SJNNV)





aSamples taken after 1 hour virus adsorption


bTimes after SJNNV infection


cTimes after RGNNV superinfection


dTimes after RGNNV infection


eTimes after SJNNV superinfection






At the times indicated in Table 3, both cells and supernatants were collected from 2 wells of each experimental group. Negative controls were collected at the end of the experiment. Total RNA was extracted from the cells using Trizol®, as described in Example 9. cDNA was synthesized using the SuperScriptTM II First-Strand Synthesis System for RT-PCR (Invitrogen), following manufacturer's instructions and adding random hexamers (50 ng) and 1 μg of total RNA in each reaction. Viral genomes were quantified using SYBR Green I-based absolute real-time PCR (qPCR), and the data were analyzed, also as described in Example 9.
To determine viral titers, several cell-free supernatants were titrated to determine TCID50 (Example 1). In groups 3 to 5, neutralization assays were performed before viral titration using the following polyclonal antibodies (1/100 dilution in L-15 medium, supplemented with 1% penicillin-streptomycin): (i) anti-NNV ab26812 (Abcam), that neutralizes the RGNNV genotype, and (ii) an anti-SJNNV antibody developed in rabbit (Dr T. Nakai, University of Hiroshima, Japan), that neutralizes SJNNV.
FIG. 7 shows measurements of SJNNV RNA 2 copy number in infected cells. FIG. 7A shows SJNNV copy number in the SJNNV+RGNNV coinfection group (Group 3), compared to SJNNV copy number in the SJNNV-infected control group (Group 1). FIG. 7B shows SJNNV copy number in the SJNNV infection, 24 hours later RGNNV coinfection group (Group 4), compared to copy number in the SJNNV-infected control group (Group 1). FIG. 7C shows SJNNV copy number in the RGNNV infection, 24 hours later SJNNV superinfection group (Group 5), compared to the copy number in the SJNNV-infected control group (Group 1). Bars show the standard deviation of two different samples. Statistically significant changes (p<0.01) are represented by an asterisk.
FIG. 8 shows measurements of RGNNV RNA 2 copy number in infected cells. FIG. 8A shows RGNNV copy number in the SJNNV+RGNNV coinfection group (Group 3), compared to RGNNV copy number in the RGNNV-infected control group (Group 2). FIG. 8B shows RGNNV copy number in the SJNNV infection, 24 hours later RGNNV coinfection group (Group 4), compared to copy number in the RGNNV-infected control group (Group 2). FIG. 8C shows RGNNV copy number in the RGNNV infection, 24 hours later SJNNV superinfection group (Group 5), compared to the copy number in the RGNNV-infected control group (Group 2). Bars show the standard deviation of two different samples. Statistically significant changes (p<0.01) are represented by an asterisk.
The data from these experiments indicated that, in cells infected with SJNNV and RGNNV: i) SJNNV genome replication was partially inhibited in the presence of RGNNV; ii) RGNNV genome replication was stimulated in the presence of SJNNV, and iii) these effects on genome replication did not correlate with production of infectious virus.
The data indicating inhibition of SJNNV replication by RGNNV is described as follows. In the coinfection group (Group 3), the data (FIG. 7A) showed a decrease in SJNNV genome copy number, as compared to the control group (Group 1), at the 12 and 48 hour time points. This was more apparent in the experiment where RGNNV-infected cells were superinfected 24 hours later with SJNNV (Group 5). In that experiment, the data (FIG. 7C) showed a decrease in SJNNV genome copy number, as compared to the control group (Group 1), at the 0, 12, 24, 48, 72 and 96 hour time points. These decreases in SJNNV genome copy number were not clearly correlated with titers of SJNNV virus produced by the cells.
The data indicating stimulation of RGNNV replication by SJNNV is described as follows. In the coinfection group (Group 3), the data (FIG. 8A) showed an increase in RGNNV genome copy number, as compared to the control group (Group 2), at the 24, 48 and 72 hour time points. This was more apparent in the experiment where SJNNV-infected cells were superinfected 24 hours later with RGNNV (Group 4). In that experiment, the data (FIG. 8B) showed an increase in RGNNV genome copy number, as compared to the control group (Group 2), at the 0, 12, 24 and 48 hour time points. However, at 96 hours, RGNNV genome copy number was decreased as compared to the control group. The increases in RGNNV genome copy number were not clearly correlated with titers of RGNNV virus produced by the cells.
We have also found that infection of cells with SJNNV, prior to or approximately simultaneous with RGNNV infection, at least initially stimulates replication of RGNNV RNA in the cells, as compared to replication of RGNNV in cells that do not contain SJNNV. This effect on replication may not correlate with infectious virus produced by the infected cells.
We have also found that infection of cells with RGNNV, prior to or approximately simultaneous with SJNNV infection, partially inhibits replication of SJNNV RNA in the cells, as compared to replication of SJNNV in cells that do not contain RGNNV. This effect on replication may not correlate with infectious virus produced by the infected cells.
While example compositions, methods, and so on have been illustrated by description, and while the descriptions are in considerable detail, it is not the intention of the applicants to restrict or in any way limit the scope of the application. It is, of course, not possible to describe every conceivable combination of components or methodologies for purposes of describing the compositions, methods, and so on described herein. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the disclosure is not limited to the specific details, the representative apparatus, and illustrative examples shown and described. Thus, this application is intended to embrace alterations, modifications, and variations that fall within the scope of the application. Furthermore, the preceding description is not meant to limit the scope of the invention. Rather, the scope of the invention is to be determined by the appended claims and their equivalents.
REFERENCES


    Iwamoto T, Nakai T, Mori K, Arimoto M, Furusawa I “Cloning of the fish cell line SSN-1 for piscine nodaviruses”. Dis Aquat Organ. 2000 November 14; 43(2):81-9.
    Iwamoto et al “Establishment of an infectious RNA transcription system for the Striped Jack nervous necrosis virus, the type species of the betanodaviruses” Journal. of General Virology (2001), 82, 2653-2662
    Lopez-Jimena B, Alonso Mdel C, Thompson K D, Adams A, Infante C, Castro D, Borrego J J, “Tissue distribution of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) genome in experimentally infected juvenile European seabass (Dicentrarchus labrax).” Vet Microbiol. 2011 December 29; 154(1-2):86-95
    Mori, K, Mangyoku, T, Iwamoto, T, Arimoto, M, Tanaka, Si, Nakai, T “Serological relationships among genotypic variants of betanodavirus” Diseases of Aquatic Organisms Vol. 57 no.1-2 page.19-26 (20031203)
    Nishizawa T, Mori K, Furuhashi M, Nakai T, Furusawa I, Muroga K. “Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish” J Gen Virol. 1995 July; 76 (Pt 7):1563-9.
    NISHIZAWA, M. FURUHASHI, T. NAGAI, T. NAKAI, AND K. MUROGA “Genomic Classification of Fish Nodaviruses by Molecular Phylogenetic Analysis of the Coat Protein Gene” APPLIED AND ENVIRONMENTAL MICROBIOLOGY, April 1997, p. 1633-1636
    Olveira J G, Souto S, Dopazo C P, Thiéry R, Barja J L, Bandin I. “Comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment.” J Gen Virol. 2009 December; 90(Pt 12):2940-51
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## Claims

1. Vaccine comprising striped jack nervous necrosis virus (SJNNV), wherein the vaccine protects a fish against red-spotted grouper nervous necrosis virus (RGNNV) infection;
wherein the fish is selected from the group consisting of European sea bass, Senegalese sole, gilt-head sea bream, red-banded seabream, common seabream, white seabream and wild meager; and
wherein SJNNV is in the form of a heat killed or chemically inactivated SJNNV.

2. Vaccine as claimed in claim 1, wherein the SJNNV is SJNNV with a cDNA nucleotide sequence which is at least 95% identical with the nucleotide sequence of nucleotides 604 to 1030 of SEQ ID NO: 4.

3. Vaccine as claimed in claim 1, wherein the SJNNV is SJNNV with an amino acid sequence which is at least 95% identical with amino acid sequence of amino acids 204 to 331 SEQ ID NO: 5.

4. Vaccine as claimed in claim 1, wherein SJNNV is in an amount of at least about 1×104 TCID50/dose.

5. Vaccine as claimed in claim 1, wherein the SJNNV comprises a SJNNV RNA2 having a cDNA sequence of SEQ ID NO: 4.

6. Vaccine as claimed in claim 1, wherein chemically inactivated SJNNV is inactivated using binary ethyleneimine.

7. Vaccine comprising SJNNV nucleic acids; wherein the SJNNV nucleic acids are in the form of a DNA vaccine; and
wherein the SJNNV nucleic acids comprise a cDNA sequence at least 95% identical with the nucleotide sequence of nucleotides 604 to 1030 of SEQ ID NO: 4.

8. Vaccine as claimed in claim 5, wherein the SJNNV further comprises a SJNNV RNA1 having a cDNA sequence of SEQ ID NO: 1.

9. A method for protecting fish against disease associated with red-spotted grouper nervous necrosis virus (RGNNV) infection, comprising administering an inactivated striped jack nervous necrosis virus (SJNNV) vaccine to a fish in need of protection from RGNNV infection;
wherein the fish is selected from the group consisting of European sea bass, Senegalese sole, gilt-head sea bream, red-banded seabream, common seabream, white seabream and wild meager.

10. The method of claim 9, where the SJNNV vaccine comprises heat killed or chemically inactivated SJNNV comprising a SJNNV RNA1 having a cDNA sequence of SEQ ID NO: 1 and a SJNNV RNA2 having a cDNA sequence of SEQ ID NO: 4.

11. The method of claim 9, where the administering of the SJNNV vaccine is performed by intramuscular or peritoneal injection of the fish with the vaccine or immersion of fish in a bath containing the vaccine.

12. The method of claim 9, where an amount of the SJNNV administered to the fish is between about 1×104 and about 1.5×104 TCID50/fish.

13. The method of claim 9, where the administering of the SJNNV vaccine results in increased transcriptional expression of at least one interferon-inducible gene.

14. The method of claim 13, where the interferon-inducible gene includes Mx.

15. The method of claim 9, wherein the SJNNV vaccine is a DNA vaccine comprising a cDNA sequence at least 95% identical with nucleotides 604 to 1030 of SEQ ID NO: 4.