Patent Application
20180147274
The present invention relates to further characterisation of the genetic stability of infectious pancreatic necrosis virus (IPNV), in particular to a greater appreciation and understanding of the frequency of mutagenesis and codon interactions that influence the replication capabilities, virulence and immunogenicity of the virus. Thus, a method for developing a commercially applicable IPNV vaccine and a vaccine comprising the IPNV genome carrying the particular mutations and codons for use in prophylaxis or treatment of infectious pancreatic necrosis (IPN) disease are also part of the present invention.
RNA viruses have large genetic diversity and high mutation rates (Steinhauer & Holland 1987). Several biological aspects can be put in the context of these genetic variations, for example virulence, persistence and adaptation both in vitro and in vivo. It is well documented that replication of IPNV relies on a RNA-dependent RNA polymerase which notably lacks the proof reading capabilities of DNA polymerases associated with 3′-5′ exonuclease activity (Duarte et al., 1994; Kohlstaedt et al., 2009; Steinhauser et al., 1992). This natural impediment of the virus results in genetic mutation rates of up to 1 mutation per 103 bases copied per replication cycle (Drake and Holland, 1999).
In addition and as a further natural deficiency of RNA genome viruses such as IPNV, they have no post-replication error corrections, which gives them low replication fidelity and the possibility of rapid emergence of mutant virus strains (1)—(Domingo, E., C. Escarmis, et al., 1996). These mutations result in a cloud or a population of closely related sequence variants within a host (or several hosts) and can differ by as little as one nucleotide from the average sequence in the population (3)—(Lauring, A. S., and R. Andino 2010). Massive viral infections often results in increased fitness and virulence (1), and movement in the mathematical fitness “landscape”, where new variants will increase to more virulent variants in the virus population (7)—(Wright, S. 1931).
The underlying biological implications of these phenomena have been clearly demonstrated for many viruses like human immunodeficiency virus-1 (HIV), hepatitis C virus (HCV), polio and other virus species. The propensity to mutate is a major challenge for the development of safe live, attenuated RNA virus vaccines.
The IPNV genome consists of two segments of double-stranded RNA that are surrounded by a single-shelled icosahedral capsid of 60 nm in diameter. Genomic segment A (typically 3097 nucleotides) encodes a 106 kDa precursor polyprotein composed of pVP2-VP4-VP3, in that order, and a 15 kDa non-structural VP5 protein, found only in infected cells. Segment B (typically 2777 nucleotides) encodes a minor internal polypeptide VP1 (94 kDa), which is the virion-associated RNA-dependent RNA polymerase (RdRp).
VP2 is a major viral capsid protein and it has been hypothesised that variations in the amino acid residues of this protein may be associated with changes in virulence. In fact, by a comparison of the amino acid sequences of various field isolates exhibiting different mortality in Atlantic salmon fry, the putative motifs involved in virulence of IPNV strains have been proposed. By way of example, such strains typically have residues threonine, alanine, threonine/alanine, and tyrosine/histidine at positions 217, 221, 247 and 500 of the VP2 sequence. Further work has shown that virulent isolates possess residues Thr217 and Ala221; moderate to low virulent strains have Pro217 and Ala221; and strains containing Thr221 are almost always avirulent, irrespective of the residue at position 217.
RNA viruses such as IPNV and IBDV are prone to change through a variety of mechanisms, so it has been assumed that there always is a risk that the virus will revert to greater virulence during multiplication in the vaccinated fish. Such a strategy would require vigilance in monitoring field viruses and the natural history of the disease. So far, unawareness about factors influencing the virulence and genetic stability of IPNV has precluded the use of avirulent strains for vaccine purposes.
Further characterization of the underlying pressures which affect the genetic stability of infectious pancreatic necrosis virus (IPNV) and the frequency of mutagenesis and codon interactions that influence the replication capabilities, virulence and immunogenicity of the virus is much needed for developing suitable avirulent but at the same time immunogenic IPNV vaccines.
The present invention whichhas been described below addresses some of the above identified problems.
The present invention provides novel genetic variants of infectious pancreatic necrosis virus (IPNV), in particular variants with greater genome stability which can be used as vaccines in prophylaxis or treatment of infectious pancreatic necrosis (IPN) disease.
It was surpisingly identified by the present inentors that the occurance of certain nucleotides at particular positions in a codon were able to confer a preferred selection of other particular nuclelotides at other particular positions in a codon and thus select for avirulent IPNV which eshibit greater genetic stability. Genetic stability of IPNV is relevant if there are to be selected suitable virla strains for commercial exploitation such that they do not revert to a virulent state. These observations allowed the present inventors to arrive at the present invention. This level of assessment has not been performed prior to this invention.
With the purpose to study the genetic stability of IPNV, different variants made by reverse genetics (clones of virus) were used to establish a persistent infection in Atlantic salmon fry. The strains used represented different virulence categories defined by amino acid position 217 and 221 of the VP2 protein of the virus. A moderately virulent strain carried P217A221, while an avirulent strain had P217T221. These strains were used to infect fry and the strains were also combined in the same inoculum (a mixture).
Accordingly, one aspect of the present invention relates to a live avirulent IPNV variant which does not revert to a virulent virus after at least 3 passages or at least 6 passages. According to one embodiment, the live avirulent infectious pancreatic necrosis virus (IPNV) do not revert to a virulent virus after at least 9 passages.
One embodiment relates to a live avirulent IPNV according to the present invention, which when delivered by immersion at a titre of 5×104 TCID50/ml to Atlantic salmon fry held in fresh water at a temperature of 12° C. causes the fry to be virus positive measured by reisolation on RTG-2 cells.
One embodiment relates to a live avirulent IPNV according to the present invention, which when delivered by immersion at a titre of 5×104 TCID50/ml to Atlantic salmon fry held in fresh water at a temperature of 12° C. provides the fry with protection against IPN disease.
One embodiment relates to a live avirulent IPNV according to the present invention, which when delivered by immersion at a titre of 5×104 TCID50/ml to Atlantic salmon fry held in fresh water at a temperature of 12° C. does not cause the fry to develop signs of IPN disease.
In one preferred embodiment, said live avirulent IPNV comprises a nucleic acid encoding a VP2 protein (SEQ ID NO: 2), wherein the amino acid in position 221 of the VP2 protein is Val and not Ala.
In one embodiment, said live avirulent IPNV incorporates a codon at position 220 of the nucleotide sequence coding for VP2 (SEQ ID NO: 3) that is associated with hypervariable codons 217, 221, and 247 of SEQ ID No: 2.
In one further embodiment, said live avirulent IPNV incorporates at position 3 of codon 220 (codon 220.3) is associated with a statistically significant interaction value with that at position 1 of nucleotide codons 247, 217 and position 3 of nucleotide codon 221 (221.3).
In one further embodiment, said avirulent IPNV, the presence of G (guanine) or A (adenosine) at position 3 of codon 220 (codon 220.3) is associated with a statistically significant interaction value with position 1 of nucleotide codons 247 and 217, and position 3 of nucleotide codon 221 (221.3).
In one yet furthe rembodiment, said live avirulent IPNV incorporates a combination of codones selected from T217A221T247.
In one preferred embodiment said codons cause the avirulent INPV not to revert to a virulent virus after at least 3 passages.
One preferred embodiment relates to the live avirulent IPNV according to the present invention, which when delivered by immersion at a titre of 5×104 TCID5o/ml to Atlantic salmon fry held in fresh water at a temperature of 12° C.
According to a second aspect the present invention relates to a method of identifying an avirulent IPNV, the method comprising the steps of:
A second aspect the present invention relates to a method of identifying an avirulent IPNV, the method comprising the steps of:
According to one embodiment of the second aspect, there is provided a method of identifying an avirulent IPNV, the method comprising the steps of:
A third aspect of the present invention relates to a live avirulent IPNV according to the present invention or to the IPNV obtained from the method of the present invention, for use as a vaccine.
A fourth aspect of the present invention relates to a live avirulent IPNV according to the present invention, the IPNV obtained from the process of the present invention or a vaccine according to the present invention for use as a vaccine against IPN disease.
A fifth aspect of the present invention relates to a live avirulent IPNV according to the present invention, the IPNV obtained from the method of the present invention or a vaccine according to the present invention for use as a vaccine against IPN disease, wherein distribution is by immersion, oral administration or injection.
Preferred embodiments of the present invention are depicted in the dependent claims and in the detailed description of the invention.
Preferred embodiments of the present invention will now be illustrated in more detail with reference to the accompanying figures.
An ideal vaccine for IPNV should induce long lasting protection at an early age, prevent virulent carrier formation and be effective against a large number of IPNV serotypes. Injection cannot be used for small fish, therefore either oral delivery or immersion are most preferred routes for early vaccination. In addition, it must be genetically stable such that it does not revert to a virulent viral strain before it can facilitate the development of an immune response to subsequent IPNV exposure.
Previously it has been assumed that these attributes of an ideal IPNV vaccine must be met either by a recombinant subunit vaccine or by an inactivated viral vaccine, as a live avirulent vaccine could potentially lead to virulent carrier formation and disease in case the avirulent virus should revert to a virulent virus. In this regard, the present inventors have alse reported the development of an avirulent IPNV strain which induces long lasting protection at an early age, prevents virulent carrier formation, is effective against a large number of IPNV serotypes and may be delivered by, injection, oral administration or by immersion, preferably the latter (G700 strain which was deposited under ECACC-No. 11 041201; WO2012/078051 is incorporated by reference in its entirety for all purposes). Interestingly, the fish infected with the G700 strain seemed to be protected from developing IPN when mixed with fish carrying the virulent strain. These findings revealed a potential for this field strain as a live avirulent vaccine against IPN.
In order to further characteris the underlying genetic mechanisms which influence the genetic stability of IPNV avirulent strains, the present inventors chose to stury this in greater detail by using a combination of statistical and in vivo correlation experiments to try and understand whether there are particular regions within the viral genome e.g. within the VP2 protein, which would affect or otherwise contribute to the preferential selection of a particular genotype over another.
In aprticular they found that some codones within the VP2 gene if already selected would influence other sequences such that a more avirulent or less virulent IPNV strain is eventualy propagated using their methods.
In the context of the invention, the term “reconstructed” or “recombinant” in relation to a sequence, nucleic acid, indicates a sequence, nucleic acid or unit which does not exist naturally in the virus and has been assembled and/or inserted in said virus or an ancestor thereof, using recombinant DNA technology, (also called gene cloning or molecular cloning) which refers to techniques of transfer of DNA from one organism to another.
The term “recombinant” in relation to a IPNV refers to an existing IPNV whose genome has been modified by insertion of at least one heterologous nucleic acid sequence, i.e., DNA which corresponds to a gene or part thereof not identical to the nucleic acid sequence of a gene naturally present in the existing virus.
Alternatively, the existing IPNV may be genetically modified by the incorporation into the virus genome of a homologous nucleic acid sequence, i.e., DNA which corresponds to a gene or part thereof identical of a gene naturally present in existing IPN virus. It will be understood that the resulting recombinant IPN virus can be manufactured by a variety of methods, and once made, can be reproduced without use of further recombinant DNA technology.
In the present description, the terms “nucleic acid” “nucleic sequence” and “nucleotide sequence” are used interchangeably and refer to a sequence of deoxyribonucleotides and/or ribonucleotides. The nucleotide sequence may be first prepared by e.g., recombinant, enzymatic and/or chemical techniques, and subsequently replicated in a host cell or an in vitro system. The nucleotide sequence preferentially comprises an open reading frame encoding a peptide. The nucleotide sequence may contain additional sequences such as a transcription terminator, a signal peptide, an IRES, an intron, etc. Preferably, the gene does not contain an intron.
The term “heterologous nucleic acid sequence” refers to a DNA which corresponds to a gene or part thereof not identical to the nucleic acid sequence of a gene naturally present in IPN virus particularly to G700 strain(deposited under ECACC-No. 11 041201; WO02012/078051 is incorporated by reference in its entirety for all purposes). The term “homologous nucleic acid sequence” refers to a DNA which corresponds to a gene or part thereof identical of a gene naturally present in IPNV.
The term “vaccine” as used herein includes an agent which may be used to cause, stimulate or amplify the immune system.
In a first aspect of the present invention relates to a live avirulent infectious pancreatic necrosis virus (IPNV) which preferably do not revert to a virulent virus after at least 3 passages or after at least 6 passages in hosts known to be susceptible to IPNV. According to one embodiment, the live avirulent infectious pancreatic necrosis virus (IPNV) do not revert to a virulent virus after at least 9 passages.
As used herein, the term “live” as applied to viruses refers to a virus that retains the ability of infecting an appropriate host (as opposed to inactivated or subunit vaccines).
As used herein, the term “avirulent” as applied to viruses is understood to mean a virus strain which has substantially lost, preferably completely lost, its ability to cause disease in fish infected with the strain, although its ability to invade fish, i.e. to penetrate into the fish by the usual route of the virus and to reproduce in the body of the fish, remains substantially intact. It is also envisaged that the immunogenisity of the avirulent viruses would be at least maintained or imporved relative to a wilt type virus.
The term “infectious” as applied to viruses indicates that the virus has the ability to reproduce. The virus can be pathogenic or nonpathogenic and still be infectious.
The term “pancreatic necrosis virus (IPNV)” refers to the causative agent of IPN and is a member of the Genus Aquabirnavirus, family Birnaviridae.
The term “virulent” as applied to viruses herein indicates that the virus is pathogenic, meaning that the virus causes disease to its host.
The term “revert to a virulent virus” as applied to avirulent viruses refers to the process where an avirulent virus revert to a pathogenic virus (meaning that the virus cause disease to its host) by naturally occurring processes (usually a mutation in position 217 and/or 221 and/or 220 and/or 247 of the VP2 protein).
Preferably said virus does not revert to a virulent virus after 6, 8, 10, 15, 20, 25, 30, 35 or 50 passages in hosts known to be susceptible to IPNV. More preferably said virus does not revert to a virulent virus after at least 7, 9, 11, 15, 20, 25, 30, 35 or 50 passages in hosts known to be susceptible to IPNV. Most preferably said virus does not revert to a virulent virus.
In one embodiment the VP2 protein is NVI-15PA which is characterised by the amino acid sequence according to SEQ ID No: 4. In another embodiment NVI-15PA is encoded by the nucleotide sequenc of SEQ ID No: 3 or a fragment thereof.
In one further embodiment the VP2 protein is NVI-025 which is charcaterised by the amino acid sequence according to SEQ ID No: 5. In another embodiment NVI-025 is encoded by the nucleotide sequence of SEQ ID No: 9 or a fragment thereof.
In one embodiment according to the first aspect of the present invention, the host is Atlantic salmon (Salmo salar L.) fry preferably of the AquaGen breed.
If the live avirulent IPNV according to the first aspect of the present invention is delivered by immersion at a titre of 5×104 TCID50/ml to Atlantic salmon fry held in fresh water at a temperature of 12° C. it causes the fry to be virus positive measured by reisolation on RTG-2 cells. However, the salmon fry were identified to be virus negative when measured by immunohistochemistry which suggests that the virus is present in the fry only in subclinical levels.
The term “TCID50” as used herein refers to the amount of virus required to produce a cytopathic effect in 50% of inoculated tissue culture cells. Virus infection in cells is complex and results in many changes to the host cell, known collectively as the cytopathic effect (CPE). Such changes include altered shape, detachment from substrate, lysis, membrane fusion, altered membrane permeability, inclusion bodies and apoptosis. The method used to determine the TCID50 value is well known to a man skilled in the art (Beitrag zur kollektiven Behandlung pharmakologischer Reihenversueche, Kärber G., vol. 162, 1931).
Even though the virus was present in the salmon fry only in subclinical levels, the live avirulent IPNV according to the first aspect of the present invention has been shown to provide the fry with protection against IPN disease, in particular as compared to fry which have not been exposed to the live avirulent IPNV according to the first aspect of the present invention.
Further, if the live avirulent IPNV according to the first aspect of the present invention is delivered by immersion at a titre of 2×105 TCID50/ml to Atlantic salmon fry held in fresh water at a temperature of 12° C. it does not cause the fry to develop any signs of IPN disease, in particular no pathological lesions have been observed in internal organs including the pancreas of the fry (histologically).
As previously disclosed, IPNV has typically two structural proteins which form the IPNV capsid. Virus protein 2 (VP2) of IPNV is one out of these two structural proteins and has been shown to be responsible for the production of type-specific monoclonal antibodies. It has previously been suggested that the sequence of the VP2 protein decides whether the virus is virulent or avirulent, and in case of the latter it has now been shown that certain amino acids of the VP2 protein are important determinants as to whether the avirulent virus may revert to a virulent virus.
In one embodiment according to the first aspect of the present invention, said virus comprises a nucleic acid encoding a VP2 protein, wherein the amino acid in position 221 of the VP2 protein is not Ala.
In another embodiment according to the first aspect of the present invention, said virus comprises a nucleic acid encoding a VP2 protein, wherein position 3 of codon 220 is associated with a statistically significant interaction value with position 1 of nucleotide codons 247 (247.1), 217 (217.1) and position 3 of nucleotide codon 221 (221.3).
According to a further embodiment of the first aspect, said virus comprises a nucleic acid encoding a VP2 protein, wherein the presence of G (guanine) or A (adenosine) at position 3 of codon 220 (codon 220.3) is associated with a statistically significant interaction value with position 1 of nucleotide codons 247 (247.1), 217 (217.1), and position 3 of nucleotide codon 221 (221.3).
The term “statistically significant” as used in the context of the present inventino is intended to mean the likelihood that a result or relationship is caused by something other than mere random chance. Statistical testing assessments are preferably employed to determine if a result is statistically significant or not. Different staristical tests would be know to the skilled person.
According to a yet further embodiment of the first aspect, said virus comprises a nucleic acid encoding a VP2 protein, wherein the live avirulent IPNV incorporates a combination of codones selected from T217A221T247. Sequence homologues of IPNV which exhibit avirulent characteristics as contemplated by the present invention are also encompassed within the scope of the present invention.
In one preferred embodiment, said homologues exhibit sequence identity is selected from the group consisting of at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity and 100% sequence identity.
The term “sequence identity” indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. The two sequences to be compared must be aligned to best possible fit possible with the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as:
wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. A gap is counted as non-identity of the specific residue(s). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson and Lipman 1988) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST).
In one embodiment of the invention, alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al 1994, available at http://www2.ebi.ac.uk/clustalw/.
According to another aspect, the present invention provides a method for identifying an avirulent IPNV, the method comprising the steps of:
According to one embodiment of the second aspect, there is provided a method for identifying an avirulent IPNV, the method comprising the steps of:
According to a further embodiment of the second aspect there is provided a method for identifying an avirulent IPNV, where the method further comprising the steps of:
As used in the context of the present invention the term “correlating” or “correlation” is intended to mean a statistical measure that indicates the extent to which two or more variables, such as the occurrence of a nucleotide in a given position of a codons, which fluctuate together. A positive correlation indicates the extent to which those variables increase or decrease in parallel; a negative correlation indicates the extent to which one variable increases as the other decreases.
Preferably the method of the second aspect identified an avirulent IPNV which does not revert to a virulent virus after 6, 8, 10, 15, 20, 25, 30, 35 or 50 passages in hosts known to be susceptible to IPNV. More preferably said virus does not revert to a virulent virus after at least 7, 9, 11, 15, 20, 25, 30, 35 or 50 passages in hosts known to be susceptible to IPNV. Most preferably said virus does not revert to a virulent virus.
According to another aspect of the present invention there is provided a vaccine, comprising the live avirulent IPNV according to the first aspect of the present invention or an avirulent IPNV obtained by the method of the present invention.
According to another aspect of the present invention there is provided a live avirulent IPNV according to the first aspect of the present invention or an avirulent IPNV obtained by the method of the present invention, for use as a vaccine.
In one preferred embodiment according to this aspect of the present invention, the live avirulent IPNV according to the first aspect of the present invention or an avirulent IPNV obtained by the method of the present invention, is for use as a vaccine against IPN disease in fry and/or smolt and/or fish; more preferably for use as a vaccine against IPN disease in fry.
In one particularily preferred embodiment according to this aspect of the present invention, the live avirulent IPNV according to the first aspect of the present invention or an avirulent IPNV obtained by the method of the present invention is for vaccination of fry against IPN disease.
In one preferred embodiment according to this aspect of the present invention, the vaccine is distributed by immersion, oral administration or injection; more preferably distributed to fry by immersion, oral administration or injection.
In another preferred embodiment according to this aspect of the present invention, said fry are kept in an environment which is free of virulent IPNV for at least 5, 10 or 15 days post vaccination with said avirulent IPNV.
In another preferred embodiment according to this aspect of the present invention, said virus or vaccine is distributed no later than day 4 after start feeding the fry.
In another preferred embodiment according to the third aspect of the present invention, distribution is by immersion using a virus dosage in the range 1×104 TCID50/ml to 1×106 TCID50/ml; more preferably 5×104 TCID50/ml to 5×105 TCID50/ml; even more preferably 1×105 TCID50/ml to 5×105 TCID50/ml; and most preferably about 2×105 TCID50/ml.
The present invention also relates to a live avirulent IPNV for the prophylaxis or treatment of IPN disease.
In a further aspect, the present invention is directed to a method of generating an avirulent IPNV comprising the steps of:
The steps of characterising the distribution of nucleotides of the VP2 protein can involve both statistical as well in vitro and/or in vivo experimental verification and testing prosedures. These prosedures have been described in the present invention. It is also contemplated that in silico methodologies may be used in generating and characterising different avirulent strains of IPNV according to the present invention. The different in vitro, in vivo and in silico would be well know to those of skill in the art.
Having now fully described the present invention in some detail by way of illustration and example for purpose of clarity of understanding, it will be obvious to one of ordinary skill in the art that same can be performed by modifying or changing the invention by with a wide and equivalent range of conditions, formulations and other parameters thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.
The following materials and methods & results are inclided as illustrative examples on how to make and use the invention. It is not intended that these examples should limit the scope of the invention in any manner or to any degree. By way of example only the techniques described in the following publication: Molecular cloning: A laboratory manual: Edited by Sambrook, Fritsch and Maniatis—Fourth Edition: 2012 (9), may be relied upon by those aiming to carry out the claimed scope of the present invention.
Cells and Viruses
Chinock salmon embryo (cells (CHSE-214; ATCC CCL-1681) were maintained at 20° C. in L-15 medium (Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS, Medprobe), 2 mM L-glutamin (Sigma-Aldrich) and 50 □g ml−1 gentamicin (Sigma-Aldrich). Rainbow trout gonad cells (RTG-2; ATCC CCL-55) were grown at 20° C. in L-15 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamin and 50 □g ml−1 gentamicin.
All virus isolates were propagated in RTG-2 cells by inoculation of 100 μl virus stock solution (stored at −20° C. in 30% glycerol, titer 107 TCID50/ml) onto 70% confluent T-162 cm2 cell culture flasks. The supernatants were harvested when widespread cytopathic effect was visible, 5-7 days post infection. The cell culture supernatants were obtained after a centrifugation at 2500G for 10 minutes, and sterile filtration (0.22 μl). The infectious titer was determined by end point dilution on CHSE-214 cells, and the TCID50 was estimated by the method of Karber (Karber 1931).
Construction of Full-Length cDNA Clones.
Generation of full-length cDNA clones of the entire coding and non-coding regions of the different isolates of segments A and B was performed according to procedures described previously (4, 6, 8). The genetically engineered virus strains were propagated on RTG-2 monolayers. The supernatants were harvested following centrifugation at 2500G for 10 minutes and sterile filtration (0.22 μl). RNA extraction using the QIAamp viral mini kit (Qiagen) was carried out according to the manufacturer's recommendation. To ensure that the generated clones had the correct residues, the genomes of segment A and B were sequenced (8) and the chromatograms were analyzed as described (2).
Plaque Purification Assay
To generate a PT variant we took advantage of previously observed attenuation characteristics of IPNV seen in CHSE-214 following passage (6). rNVI-15PA was passaged ten times on CHSE-214 cells. The obtained isolate was then plaque purified by inoculating RTG-2 monolayers on six well plates with 10-fold dilution (10−3 to 10−8) of cell culture supernatants. After 1 hr adsorption at room temperature the inoculum was removed and the cells were overlaid with 0.8% SeaPlaque Agarose (BioWhittaker) in 2×L-15 medium (Sigma) containing 5% FBS and 1% L-glutamine. The cells were incubated at 15° C. for 4 days and plaques formed by cytopathic effect (CPE) were picked by insertion of a 3 mm punch biopsy (Miltex) through the agar to the plate.
The plaques were subsequently inoculated on RTG-2 monolayers and incubated at 15° C. until full CPE was observed. Then the supernatant was harvested following a centrifugation at 2500G for 10 minutes and sterile filtration (0.22 μl). RNA was extracted using the QIAamp Viral RNA mini kit according to the manufacturer's recommendations. Complete nucleotide sequences of segment A and B of each virus was determined as described before. Prior to challenge the isolates were propagated by one passage in RTG-2 cells.
In Vivo Challenge Experiment Atlantic Salmon Fry.
The challenge was conducted at VESO Vikan's research facility, Namsos, Norway. A total of 1020 Atlantic salmon (Salmo salar L.) fry of the AquaGen strain hatched at the VESO Vikan hatchery were included in the experiment. The fry had recently started to take feed (Micro 015, Ewos). At arrival at the research facilities, 20 fish were weighed and the average weight of the fry was 0.2 grams. The rest of the fish were divided into 4 tanks, each of 250 fry. After an acclimatization period of one week, the fry were starved one day prior to challenge. Fish were challenged by immersion with IPNV at a dose of 5×104 TCID50/ml in a total volume of 4 liters per tank. One tank was challenged with rNVI-15PA (Tank 1), one tank was challenged with rNVI-15PT (Tank 2), the third tank was challenged with a mix (50/50) of the two recombinant isolates (Tank 3), and one control tank was mock-infected by adding cell culture medium (Tank 4). The water was aerated during the challenge. After a period of 3 hours the water volume was reduced to 2 liters and normal flow was resumed. Mortality was recorded and dead fish were collected and frozen at −70° C. on daily basis. Sampling of ten fish from each tank was performed at ten days post challenge, and after this first sampling, ten fish were sampled from each tank once a month, up until six months post challenge.
Stress Exposures 6 Months Post Initial Challenge.
At six months post challenge, the remaining fish in each tank were divided into two parallels; A and B. Each of the parallels included 4 tanks with approximately 60 fish in each tank. Fish in tanks 1B-4B were subject to three stressful events during a period of one week. The stress was imposed by reducing water level to approximately ½ of the normal water level. In addition, the fish were chased for 15 minutes with a dipnet, at a moderate speed. Fish in parallels 1A-4A were not subject to any stress treatment (non-stressed).
After the stress treatment, samples were collected at the end of the stress period, then weekly for an additional 3 weeks post onset of stress (7 days between samplings). At each sampling, 12 fish from each group were sacrificed: six fish from each group were frozen at −70° C. and from six fish in each group the kidney was dissected and stored in RNAlater® (Qiagen).
Virus Re-Isolation from Persistently Infected Fish.
Fry samples stored at −70° C. without conservatives were added phosphate buffered saline (PBS) (1:5, weight/volume) and homogenized using a stomacher. 100 □l of this homogenate was transferred to 600 □l RLT buffer containing 2-mercaptoethanol (RNeasy Mini kit, Qiagen) and stored at −70° C. The rest of the homogenate was diluted 1:2 in L-15 medium supplemented with 2 mM L-glutamine and 50 □g ml−1 gentamicin. After a brief centrifugation at 2500G for 10 minutes the supernatant were inoculated onto RTG-2 cells grown in 24 well plates in final dilutions of 1% and 0.1%, and incubated for one week at 15° C. The cell culture medium from the first passage was used to infect new monolayers.
The samples were considered negative when no CPE was observed after 1-week incubation of the second passage. RNA was isolated from the fish homogenate for all negative samples on cell culture using the RNeasy Mini kit (Qiagen) in accordance with the supplier's protocol, and RT-PCR was performed to amplify a 224-bp IPNV-specific DNA fragment, as described by Taksdal et al. (2001) with minor modifications. Qiagen's OneStep RT-PCR kit was used according to the manufacturer's instructions, with 0.5 μg RNA and 15 pmol each of primers IPNV-F and IPNV-R (Table 1) in a total reaction volume of 25 μl. The cycling conditions were 60° C. for 30 min., 95° C. for 15 min., followed by 45 cycles at 94° C. for 45 s, 57° C. for 45 s, 72° C. for 1 min., and finally 72° C. for 10 min. The PCR products were separated by agarose gel electrophoresis and visualized by staining with SYBR® Safe DNA gel stain (Invitrogen).
Three controls were included for each RT-PCR run: one positive and one negative tissue sample, and one negative control in which water was substituted for RNA.
Sequencing
RNA was isolated from homogenate stored on RLT buffer containing 2-mercaptoethanol (RNeasy Mini kit, Qiagen) stored at −70° C. after homogensation. RT-PCR was performed to amplify a IPNV-specific fragment (of VP2) using Qiagen's OneStep RT-PCR kit according to the manufacturer's instructions, with 0.5 μg RNA and 15 pmol each of primer A-Sp500F and A-Sp1689R (Table 1) in a total reaction volume of 25 μl. The cycling conditions were 50° C. for 30 min., 95° C. for 15 min., followed by 40 cycles at 94° C. for 45 s, 57° C. for 45 s, 72° C. for 2 min.15 s, and finally 72° C. for 10 min. The PCR products were separated by agarose gel electrophoresis and analyzed by staining with SYBR® Safe DNA gel stain.
To purify the DNA fragments from agarose gel, the Quantum Prep Freeze N' Squeeze DNA Gel Extraction Spin Column (BIO-RAD) was used according to the manufacturer's instructions. The recovered DNA was sequenced by a commercial sequencing service (Eurofins MWG operon) using primer A-Sp500F (Table 1). The sequence data were analyzed using VectorNTI software (Invitrogen). The chromatograms were examined as described previously (2) and briefly, by examining individual chromatograms obtained after sequencing (see below). The computer mouse was used to run over the chromatogram and thereby we obtained a relative level of the (different) nucleotides at the different positions. A threshold of 2% (of total nucleotides in each position) was set to allow exclusion of nucleotides present at very low levels.
Cloning and Nucleotide Sequencing.
Four samples with different mixtures of bases in position 217 and 221 were picked for cloning and sequencing. Taq polymerase-amplified PCR products from the sequencing step were cloned into a TOPO® Vector by using TOPO TA Cloning® according to the manufacturers' instructions (Invitrogen). The One Shot® Chemically Competent E. coli cells were transformed according to the protocol and 50 μl were spread on prewarmed agar plates containing 50 μg/ml ampicillin and pre-incubated for 30 min at 37° C. with 40 μl 40 mg/ml X-gal (Invitrogen) in dimethylformamide for white blue selection. 80-96 white colonies were picked from each Petri dish and placed one in each well on a 96 well agar plate with 150 mg apicillin (GATC Biotech). Plasmid were isolated and sequenced by a commercial sequencing company, GATC Biotech. The sequence data were analyzed using VectorNTI software.
The nucleotide sequence of the VP2 gene is presented in SEQ ID NO: 1 and the amino acid sequence of the VP2 protein is represented by amino acid residue 1-442 of SEQ ID NO: 2.
Combination of plasmids pUC19NVI15VP2 and pUC19NVI15B resulted in the recovery of NVI-15PA (6). Ten times passage of NVI-15PA in CHSE-214 cell lines resulted in a mutation in position 221 in VP2 of NVI-15PA, A221T, and after plaque purification the rNVI-15PT isolate was recovered. The genomic RNAs of the recovered viruses were analyzed after RT-PCR amplification, and the sequence analysis of the RT-PCR products confirmed the expected mutations in the VP2 and VP1 regions of the chimeric and reassortant viruses. Furthermore, complete nucleotide sequences of segment A of both viruses were determined, and positions 252, 281, 282 and 319 had the following amino acids, VTNA, respectively (NVI-015PA is represented by SEQ ID No: 4).
The challenge dose used in this study was lower than what is used in standard challenge studies (5), since the purpose was to establish a persistent infection and to retain a high number of surviving fish. Of all the sampled fish during the experiment 94% were persistently infected. The cumulative mortality was 11% (PA), 11% (PA/PT) and 9.8% (PT) over the study period.
To document that the fish were actually infected and remained persistently infected we collected fry every 30 days from month 1-6 post infection. At each time point 10 fish were examined using cell culture and RT-PCR. Overall IPNV were reisolated by culture or detected by RT-PCR in 94% of the three groups (data not shown). All fish were positive by 6 months post challenge. Fish that were negative by culture were examined by RT-PCR. Progression from acute infection to persistence has been associated with increase in virus genome complexity. With the purpose to determine any complexity of the virus genomes of IPNV strains used here over the persistence period, RNA isolated from each of 5 fish from each group at all sampling points was amplified by RT-PCR and PCR-products were purified by gel electrophoresis and sent for sequencing.
The PA strain mutated in vitro (during initial culture) and appeared with a mutation in position 221; A221V (alanine exchanged with valine). This was not discovered prior to challenge and the “mixed” strain was thus used for challenge, meaning that the PA and PA/PT groups were challenged with a PA/PV and PA/PT/PV combination, respectively. The majority of mutations were found in residues 217, 221 and 247 (described earlier as hot spots for mutations in the VP2 of IPNV, Sp (5)). In addition positions 252, 281, 282 and 319 were examined for stability.
The variability in positions 217, 221 and 247 is described separately. The nucleotide/amino acid variation is based on examination of chromatograms after sequencing (as described above). The relative “amount” of nucleotides (and the corresponding) amino acids in each position was then entered into a “self-made” frequency calculation program that allowed comparison between the different strains and fish at the different positions, focusing on positions 217, 221 and 247 of VP2 (
When fish infected with combinations of PA/PT (actually PA/PV/PT), a similar pictures is seen with increasing variability over time post challenge (not shown).
In addition to positions 217 and 221, there is also a marked variation in position 247 with variability increasing over time post challenge.
From the results presented above, non (PT)- or low-virulent (PA) virus strains used as single or combined source of infection of salmon fry result in increasing codon and amino acid variability over time post challenge, showing the instability of the virus genome during replication in a complex environment. Positions 217, 221 and 247 show particularly high variation with numerous combinations.
From the results presented it is difficult to observer or identify co-variation or correlation between mutations in the different codons. The inventors therefore examined interactions between nucleotide positions and also held the variation against mutations (synonymous and non-synonymous mutations) in other codons.
Codon 220 was found to show variation with links to the hypervariable codons 217, 221, and 247. A 3-level hierarchic statistical model was used to analyse the interactions between codons. There is a correlation between mutations in position 220.3 and mutations in certain positions in codons 217, 221, and 247. The specific interactions are depicted below and the following positions were found to correlate with position 220.3:
The next step was to analyze if particular nucleotides in the indicated positions interact or dictates changes in other positions. We found that G and A in position 3 of codon 220 (220.3) give a highly significant statistical “interaction” value with 247.1 and 217.1, and these again impact on position 221.3. Functionally 220.3-G mutates to 220.3-A prior to changes in any of the other positions. These nucleotides (220.3, 247.1, 217.1 and 221.3) are “mixed” together in a very complicated pattern. The mathematic analysis gives the following;
Call: 1 m(formula=X220.3˜X217.1+X217.2+X217.3+X221.1+X221.2+X221.3+factor(Base)−1+Stress)
Residuals:
Coefficients:
<2e−16 ***
Further to this, the results show that stress is not a significant determinant for the interactions and mutations seen in these positions.
Residues 252, 281, 282 and 319 remained non-mutated through out the study with a preserved combination of VTNA as in the infecting virus strains (NVI-15PA is represented by SEQ ID No: 4 and NVI-025 is represented by SEQ ID No: 5).
The findings here show that the IPN virus genome is highly unstable and readily mutates post infection in vivo. There are complex interactions between various codons and with positions 217, 221 and 247 being highly variable, where synonymous mutations in the 3rd position of codon 220 impacts on the variability in the three defined codons. One interesting observation is that for both PA and PT infected fish, a combination of T217A221T247 is found as a dominating virus clone.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1505310.1 | Mar 2015 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2016/050797 | 3/22/2016 | WO | 00 |
