EP3166621A1 - Agent with antiviral properties for preventing or treating individuals exposed to a virus of the birnaviridae family - Google Patents
Agent with antiviral properties for preventing or treating individuals exposed to a virus of the birnaviridae familyInfo
- Publication number
- EP3166621A1 EP3166621A1 EP14896988.4A EP14896988A EP3166621A1 EP 3166621 A1 EP3166621 A1 EP 3166621A1 EP 14896988 A EP14896988 A EP 14896988A EP 3166621 A1 EP3166621 A1 EP 3166621A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- virus
- prophylactic
- ipnv
- composition
- peptides
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
Definitions
- the present invention relates to molecules that can be used as a prophylactic agent, therapeutic agent, a prophylactic food supplement with therapeutic potential, or an agent with antiviral properties which prevent infections or treat animals or fish that have been exposed to or are infected with a virus of the Birnaviridae family.
- the invention pertains to peptides that are effective in decreasing viral infection by reducing or impeding the production of viral particles, and can therefore prevent the potential mortalities associated with virus infection.
- Birnaviruses corresponds to a group of viruses possessing two segments of double-stranded RNA and belongs in group III according to the general classification of viruses. This group contain eight families, all of which have icosahedral symmetry, with a diameter of 60 nm and are "unwrapped". Present inside the virions are 5 proteins and between 3 and 4 peptides.
- the Birnaviridae family consisits of the Aquabirnavirus, Avibirnavirus and Entomobirnavirus genera.
- the Aquabirnavirus genus consists of birnavirus that infect fish, molluscs, crustaceans and rotifers.
- Species of the Aquabirnavirus genus are the causative agents of infectious pancreatic necrotic virus (IPNv), as well as the yellow tail ascites virus (YTAV), marine birnavirus (MABV).
- the Blosnavirus family includes the blotched snakehead virus (BSNV); the Avibirnavirus is a genus of viruses that affect specifically poultry, and corresponds to the cause of the Infectious Bursal Disease, which has great impact in poultry industry.
- the virus is Infectious Bursal Disease virus (IBDv); and finally, the Drosophila X virus is part of the Entomobirnavirus family and infects Drosophila melanogaster (fruit fly).
- the infectious pancreatic necrosis virus (IPNv) is used as a prototype birnavirus to generate a generic alternative prophylactic that can be used against the whole Birnaviridae family of viruses. Therefore, the description provided in here below directed specifically to IPNv should be understood as an example and not to be used to limit the scope of the invention. In a particular case, without intending to limit the scope of the present invention to a particular virus species, described below is one of many instances where a Birnaviridae family virus represents a major problem for a particular industry. This description should only be interpreted as an exemplification of one of the problem cases, bearing in mind that there are other members of the Birnaviridae family affecting other industries and these industries could also benefit from the present invention.
- This pathogenic agent is spread throughout all salmon producing countries and has a particular characteristic: it is latent in bottom sediments infecting different mollusks and native fish surrounding salmon farms without causing disease and can be isolated from fish farms in lakes.
- the largest outbreaks of disease occur during the early stages of development, i.e. first feeding stage and in the transfer of the fish smolt to the ocean producing outbreaks with up to 70% mortality.
- the aquatic Birnavirus are those with the greatest range of infection, infecting numerous fish species, among which are the Salmonids.
- the prototype of the aquatic Birnaviridae family is the Infectious Pancreatic Necrosis Virus (IPNv), considering members of this group, all are bisegmented double-stranded RNA viruses (dsRNA) able to cause clinical infection in one of the respective species.
- IPNv Infectious Pancreatic Necrosis Virus
- the first pathogenic birnavirus was isolated from a fish found in a brook trout (Salvelinus fontinalis) in the National Fish Hatchery, Leetown, West Virginia, USA. The agent was obtained during an epizootic of trout fingerlings suffering pancreatic necrotic infection (IPN).
- This necrotic pancreatic virus (IPNv) was deposited in the American Type Culture Center (ATCC) as ATCC VR299 IPNv and has been detected for many years in various locations in North America associated with high mortality rates in
- birnaviruses are antigenically related, representing a major serogroup (A) with 10 serotypes and only a few birnavirus not antigenically related forming a second serogroup (B).
- Most of the IPNv isolated in the USA belong to serotype A1 (West Buxton); the Canadian isolates (C1 , C2, C3, Jasper) to serotypes A6-A9 and the European isolates (Sp, Ab, I and Te) to serotypes A2-A5 and serotype A10.
- Serotypes A1 , A2 and A3 have been detected in Asia..
- the aquatic birnaviruses have similarities in morphology and biochemical and biophysical properties.
- IPNv virions present the typical characteristics of the BirnaviridaefamWy.
- the IPNv genome consists of two segments of double stranded RNA (dsRNA) of 3.1 kb (segment A) and 2.9 kb (segment B) with non-coding regions (UTR) at the 5 'and 3'.
- Segment A contains two partially overlapping open reading frames (ORFs).
- the first encodes a non-structural protein VP5 (145 aa, 17 kDa) which is a cytolytic membrane protein that while dispensable for virus replication in cell culture, is important in pathogenesis in vivo as it is involved in the release and dispersion of the viral progeny. It has been proposed that this protein inhibits apoptosis in the early stages of infection.
- VP5 145 aa, 17 kDa
- the second ORF encodes a polyprotein of 107 kDa (972 aa) which is proteolytically autoprocessed leading to the pVP2 protein (508 aa, 54 kDa ), VP4 (225 aa, 25 kDa) and VP3 (237 aa, 28 kDa) in a co-translational process mediated by VP4 itself.
- pVP2 is the precursor form of the capsid structural protein, VP2.
- the 74 carboxy terminal residues of pVP2 are processed to yield the mature form VP2 (442 aa, 48kDa).
- the maturation process requires assembly of the viral capsid and the small polypeptides generated are retained in the viral particles. Such peptides have the ability to disrupt membranes. As occurs in morphogenetic processes in other viral systems, this proteolytic maturation process could confer irreversibility to the capsid assembly.
- IPNv needs to process its own proteins to be infective. Besides possessing five major proteins including VP2, VP3 and VP4 it generates a number of peptides resulting from the processing of the polyprotein.
- cleavage sites were identified. Two of these cleavage sites (486-487, 495-496) were proposed as a target of the VP4 viral protease. Of these two sites, the primary cleavage site at the junction pVP2-VP4, is defined as a [S / T] XAA motif. This consensus sequence shows some similarity to the SKAW sequence found in the 442-443 region, suggesting that the VP4 could be involved in the cleavage to generate mature VP2.
- Apoptosis is a process of carefully regulated cell death that can be triggered by a variety of stimuli, one of these being viral infection. Some features of viral infection are recognized by cells as harmful, promoting a defensive response that ends with cell death. Induction of apoptosis in most cases is a challenge for successful viral replication. But sometimes appropriate manipulation of the apoptotic response results in a cell death mechanism which increases the development of the virus. IPNv, like many other viruses, induces apoptosis in cultured cells. This response is seen during the early stages of IPNv multiplication involving severe morphological damage to the infected cells for example, such as the blebbing or bubbling of plasmatic membrane.
- This cell death program can be interpreted as a defense mechanism of the cell against IPNv multiplication.
- most infected cells lack an apoptotic response, at least half of the infected cells do not express apoptotic signals at any time during the viral replication.
- Annexin V assay is sensitive to the earliest morphological changes produced in the cell, and can be used as an early marker of apoptosis, including apoptosis detected before the activation of the caspases.
- aquatic birnaviruses are viruses that have a great range of action, with representatives infecting numerous fish species, where salmonid fish are highlighted, for example rainbow trout (Oncorhynchus mykiss), river trout (Salvelinus fontinalis), brown trout (Salmo trutta), lake trout (Salvelinus namaycush), Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), and numerous invertebrates and marine fish such as eels (Anguilla anguilla, Anguilla japonica) striped fish, tilapia ( Tilapia mossambica), marine molluscs and crustaceans of European and Japanese coasts. Consequently, according to the regions or habitats of species referred to, IPNv is considered to be endemic in many parts of America, Europe and Asia, not being exclusive to Chile.
- IPNv is transmitted via feces, urine and sexual secretions of infected fish. For this reason it can be transmitted vertically via eggs. Studies of factors affecting the transmission and outbreaks of IPN indicate that iodophor used as disinfectant during the artificial fertilization process does not completely eradicate IPNv infectivity. The virus is also transmitted horizontally, surviving fish from IPNv outbreaks are transformed into vectors and can carry the virus throughout their lifetimes. IPNv can also be transmitted through the feces of fish-eating birds.
- the present invention is based on the use of chemically synthesized peptides for the purpose of inhibiting the assembly and / or attenuating the infectivity of IPNv in an in vitro model as well as in large scale trials.
- the document W01994004565 discloses synthetic polypeptides with antigenic properties useful in treating IPNv infections in fish, however, unlike the present invention it focuses on generating an immune response in fish, while in the present invention there is provided a prophylactic formulation which seeks to prevent transmission when an animal is exposed to the presence of one of the viruses under consideration, in particular IPNv.
- US5165925 discloses a vaccine against VR-299 strains and SP of IPNv, where the vaccine is a polypeptide segment obtained from a segment of the virus. In particular it is mentioned that this segment includes at least VP2.
- the documents US5780448 and US6180614 describe DNA vaccines where the vaccine consists of a DNA construct that encodes at least one peptide of a pathogen that attacks aquaculture species. Specific examples which comprise sequences encoding VP2, VP3, which could be combined are described.
- US6010705 discloses a vaccine based on a live attenuated virus against the microorganism Edwardsiella ictaluri. It additionally discloses that the vaccine may further comprise a coding sequence of VP2 of IPNv (claim 5).
- US20040047881 describes recombinant microorganisms which express portions of different pathogens.
- the microorganism expresses part of segment A of IPNv.
- the microorganisms that express pathogen polypeptides are used to produce food for aquatic animals.
- US20070286871 discloses a composition for preventing viral infection, where the composition generally comprises of an antigen derived from a virus and the antigen is from IPNv.
- US20070248623 describes constructs encoding various virus antigens, among which is mentioned IPNv and a DNA vaccine comprising IPNv sequences.
- US20100316663 describes a vaccine which corresponds to a fusion protein between the translocation domain of Pseudomonas aeruginosaexotoxin A and an antigenic protein of IPNv.
- US20120040010 describes a vaccine formulation comprising excipients allowing the composition to adhere to the mucous membranes of animals, within which fish are reported. It is mentioned that the vaccine can be against IPNv.
- US8168201 describes a vaccine comprising a truncated VP2 antigen of IPNv and also considers the nucleic acid sequence encoding the truncated polypeptide.
- EP1975238 discloses a vaccine for aquatic species.
- WO2002038770 describes vaccines based on polypeptides VP2 and / or VP3 of IPNv.
- WO2003015714 describes a composition which blocks viral replication (viral budding), wherein the active agent corresponds to a molecule which corresponds to the fusion of a transporter and a peptide that is part of a viral structure. It was mentioned that said viral structure may be a peptide of at least 6 amino acids, and also indicates that it can, in a particular instance, come from the IPNv VP2 protein. Nevertheless, the indication of the fusion peptide has a completely different functionality than the present invention, wherein a first peptide of the ones disclosed in WO2003015714 helps or improves the entry of a second peptide in the cell.
- WO2003013597 discloses a vaccine consisting of sub-units of the viral capsid (VP2, VP3) that form an empty capsid.
- W01999050419 discloses a method for producing a vaccine against IPNv.
- the vaccine is an attenuated virus.
- WO2008140610 describes a vaccine based on the VP2 viral capsid protein.
- the method of administration is by injection, or feeding of recombinant yeast strains that express the active agent (VP2) of the vaccine.
- WO2011138489 describes the use of casein hydrolysates as antiviral agents.
- document W01994004565 discloses synthetic peptides, where key positions are identified within peptides (R1 , R2, R3) which can be changed to different amino acids options.
- compositions of the present invention impedes the increase in viral load in chronically infected fish, or fish that are normally exposed to viral particles.
- Figure 1 Inhibition of infective units by p20.
- Semi-quantitative immune detection of IPNv in de novo infected CHSE-214 cells shows the inhibitory action of p20.
- Figure 2 Antiviral Activity of peptide p20
- A CHSE-214 cells permanently infected with IPNv, the number of copies of mRNA from VP2 of IPNv was measured with RT-qPCR from supernatant of cells.
- B Calibration curve of number of copies of Topo-VP2 IPNv for qPCR.
- Figure 3 Relative quantification of VP2 relative to elongation factor ELF. Treatment with the peptide before infection with IPNv.
- Figure 4 Relative quantification of VP2 relative to elongation factor ELF. Treatment with the peptide after infection with IPNv.
- Figure 5 Relative quantification of VP2 relative to elongation factor ELF. Treatment with peptide with alanine variations before infection with IPNV.
- Figure 6 Relative quantification of VP2 relative to elongation factor ELF. Treatment with peptide 182 with alanine variations after infection with IPNV.
- Figure 7 Change in viral titre of IPNv in CHSE-214 cells determined by focal fluorescence in a treatment with the peptide 182 before infection with IPNv.
- Figure 8 Change in viral titre of IPNv in CHSE-214 cells determined by focal fluorescence in a treatment with peptide 182 after infection with IPNv.
- Figure 9 Percentage cumulative survival after experimental trials to test effectiveness of the peptide 182 in vivo.
- Figure 10 Trend of mortality shown in trials. No mortality present after day 24 until day 56.
- Figure 11 Control versus treatment with peptide; All Results.
- Figure 12 despite the heterogeneity of viral load per fish, the presence of peptides in the food tends to lower the overall viral load, reflected in the tendency to increase the relative values of Ct in time in clear difference with the control formulation without peptide (f-5) where the trend is to maintain or even increase the average viral load (higher Ct values).
- the present invention relates to molecules with antiviral properties to prevent the spread of infection in animal or fish by a virus of the Birnaviridae family, specifically the infectious pancreatic necrosis virus (IPNv). More particularly, the invention pertains to peptides that are effective in decreasing viral infection by reducing or impeding the production of viral particles, and can therefore prevent the potential mortality resulting from virus infection and is of particular use in the prophylaxis or prevention of infections by viruses of the Birnaviridae family. In a particular embodiment, we consider the use of peptides applied to the tanks where the fish are grown or as part of a feed composition.
- IPNv infectious pancreatic necrosis virus
- the present invention pertains to synthetic peptides or fragments thereof, specially designed for the prophylactic control of viral diseases of the Birnaviridae family, more particularly the infectious pancreatic necrosis virus IPNv which affects many species of fish.
- the present invention in a first aspect, corresponds to synthetic peptides or fragments thereof having the property of reducing the rate of infection in animals exposed to a particular virus.
- the specially processed synthetic peptides were developed based on a sequence encoding pVP2 of the IPNv capsid.
- the peptides considered herein correspond to peptides with an identity of at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% compared to the amino acid sequences SEQ ID NO:1 (p182), SEQ ID NO:2 (p20), or fragments thereof.
- the peptides considered herein correspond to peptides with an identity of at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98 %, at least 99%, with respect to the nucleotide sequences SEQ ID NO:3 (p182), SEQ ID NO:4 (p20), or fragments thereof.
- the present invention comprises a prophylactic agent; therapeutic agent; prophylactic food supplement with therapeutic potential, wherein said prophylactic agent; therapeutic agent; prophylactic food supplement with therapeutic potential corresponds to at least one or both peptides described above or fragments thereof, that is, comprises at least one, two or three peptides having an identity of at least 80%, at least 85%, at least 90 %, at least 92 %, at least 95 %, at least 98 %, at least 99 % compared to the amino acid sequences SEQ ID NO:1 , SEQ ID NO:2, or fragments thereof, or at least one, two, or three peptides having an identity of at least 80%, at least 85 %, at least 90 %, at least 92 %, at least 95 %, to least 98%, at least 99 %, with respect to the nucleotide sequences SEQ ID NO:3, SEQ ID NO:4, or fragments thereof; and one or more pharmaceutically acceptable excipients.
- an application of the prophylactic formulation of the invention to animals at risk of potential contact with the source of infection of a virus of the Birnaviridae family is considered.
- the animal at risk of exposure to a virus of the Birnaviridae family is a fish.
- the virus of the Birnaviridae family is IPNv.
- the integration of the peptides of the invention in a food matrix in such a way that they can be administered or provided to growing animals is considered.
- the peptides described above are administered to farmed fish through a composite food. Since it is not possible to measure accurately the quantities of peptides that are actually consumed by each fish, ranges of amount of peptides administered in a composite food have been set.
- the peptides of the invention are in concentrations of between 10 ⁇ 10 to 10 ⁇ 5 molar (M), more preferably between 5 * 10 "9 to 10 "6 M, most preferably between 5 * 10 9 and 10 7 each one.
- the peptides of the invention when included in a food matrix may be coupled with appropriate molecules, so as to improve its absorption.
- appropriate molecules can be selected from but not limited to, polymers such as polyethylene glycol (PEG), chitosan of various molecular weight.
- the application of the prophylactic formulation of the invention is performed in a culture tank, where a dip is given to fish fry that could potentially come into contact with IPNv.
- the application of the prophylactic formulation of the present invention is applied in concentrations between 10 ⁇ 4 M to 10 ⁇ 10 M, more preferably between 2x 10 "4 M to 10 "8 M, more preferably between 8x 10 ⁇ 4 M to 10 ⁇ 6 M, even more preferably between 10 "5 to 5x 10 "5 M.
- the density of fry in the culture tank during the application of the prophylactic formulation of the invention is such to allow the proper development of the fry.
- the invention considers densities from 10 kg to 70 kg, more preferably between 15 kg and 50 kg, more preferably between 20 kg and 40 kg, more preferably between 25 and 30 kilograms of fry per cubic meter of water in the tank during application of the prophylactic formulation of the invention.
- the fry are exposed to the prophylactic formulation of the present invention for a period of between 1 and 24 hours, more preferably between 2 and 15 hours, more preferably between 4 and 10 hours, more preferably for at least 6 hours.
- the peptides were labelled with rhodamine-B, and the entry of the peptides into cells evaluated via fluorescence microscopy. Subsequently the cytotoxic capacity of the peptides were evaluated by Trypan Blue and MTT Together with this, virus expression was determined by quantitative analysis using the technique of "real-time reverse transcription PCR". In order to obtain quantitative results on the inhibition of the infectivity and to calculate viral particle titers in both infection models semiquantitative immunofluorescence will be performed.
- rhodamine-B 0.2 ml of concentration 2.5mg/ml
- CHSE-214 exponentially growing Chinook salmon embryo cells
- the culture medium was discarded and the cells were washed once with PBS (pH 7.3), subsequently rinsed three times with PBS-Tween 20 (0.05%) and analyzed under a Nikon Eclipse 400 fluorescence microscope and recorded with a Nikon Coolpix 4500 digital camera.
- CHSE-214 cells and IPNv-persistently infected cells (10, 19) were cultured as exponentially growing sub confluent monolayers on 24 well plates in MEM and Leibovitz (L-15) medium respectively, supplemented with 10% (v/v) fetal calf serum (FCS) and 2 mM glutamine and maintained. IPNv growth and titration
- the virus growth and titration were evaluated on two models of infection: fresh CHSE-214 cells infected de novo and the established CHSE-214-NV1015 cell line persistently infected with IPNv .
- fresh CHSE-214 cells infected de novo fresh CHSE-214 cells infected de novo and the established CHSE-214-NV1015 cell line persistently infected with IPNv .
- the virus propagated on CHSE-214 cell monolayers were infected at semi confluency at a multiplicity of infection of 0,001 in Leibovitz's medium (L-15, Gibco) at 18 e C, supplemented with 50 ⁇ gentamicyn, 2mM L-glutamine and 10% fetal bovine serum (FBS, Gibco).
- culture fluids were harvested and titered, quantifying the number of infective particles, using a modified Reed and Muench protocol (10, 24).
- the maintenance medium (MM) was identical except that the serum concentration was reduced to 2%.
- the plates were fixed with methanokacetone (3:1 ) for 30 min at -20°C washed and incubated with a commercial polyclonal anti VP2-VP3 (BiosChile, Chile) as the first antibody 1 /80 for 1 hour at room temperature, washed and followed by an anti-rabbit FITC-conjugated antibody (Fluorotest, BiosChile).
- the modified semi quantitative method of Reed and Muench (10, 24) was used to determine the focal fluorescent units under a fluorescent microscope.
- the distribution of the fluorescence was analyzed on a Nikon Eclipse 400 fluorescence microscope equipped with a 100-watt mercury lamp. Color photography was performed with a Nikon Coolpix 4500 digital camera.
- CHSE-214-NV1015 cells persistently infected with IPNv were grown on 24 well cell culture plates until confluence, the peptide was added at a total concentration of 10 ⁇ for four hours, washed and fresh medium added. After 24 hours of peptide treatment total RNA was extracted by the Trizol procedure and VP2 mRNAs expression measured by the RT-qPCR real time procedure (Stratagene 1 step RT-qPCR Kit) with SNPF primers 5 ' -CAA CAG GGT TCG ACA AAC CAT AC-3 ' and SNPR primer 5 ' -TTG ACG ATG TCG GCG TTT C-3 ' .
- the reaction was carried out in 30 ⁇ mixture consisting of Brilliant® II SYBR® Green QRT-PCR Master Mix Kit, 1 -Step (Stratagene, Inc.), primers and template RNA. Samples were amplified and detected using a Chromo 4 system (BioRad). The final thermo cycling profile was 50°C for 55 minutes, 94°C for 10 minutes and 94°C for 30 s, 55°C for 30 s and 72°C for 40 cycles. 800 ng of total RNA per experimental sample were used in each triplicate reaction. In order to quantify VP2 mRNA copies of IPNv, a standard curve for DNA quantification was established. To have IPNv real-time PCR standards, the VP2 region was amplified using published procedures standardized in our laboratory.
- the VP2 amplicon was cloned into PCR 2.1 vector (Invitrogen Inc.) and its specificity confirmed by sequencing. Plasmid DNA was isolated using the Quiaprep miniprep kit (Quiagen) according to the manufacturer's instructions. The purified plasmid was quantified using a Nanodrop device (Nanodrop ND-1000) and serially diluted in Sigma DNase- RNase free water to a final concentration ranging from 10 to 1 - 10 to 10 copies of genome- equivalents ⁇ L. Two microliters of each dilution were used for real-time PCR in triplicates to create a standard curve to be used to quantify putative IPNv DNA amounts in experimental samples. ( Figures 1 and 2)
- the putative toxic effect of the synthetic peptide p20 on eukaryotic cells was measured by exposing established CHSE-214 cells to the peptide according to standard procedures developed in our laboratory (5, 29). Briefly, cell monolayers at 70% semi confluence were washed with PBS and then the peptide p20 added at a range of concentrations (1 -100 mM) in triplicate wells and incubated for the maximum viability time (3 h) without culture medium. Samples were then washed three times with excess of PBS before adding 0.1 % trypsin in the presence of EDTA for 30-60 s to release cells from the monolayer. Individual cell viability was determined using the Trypan Blue exclusion technique .
- CHSE-214 cells were pretreated with decreasing molar concentration of peptide 182, from 1 * 10 to 1 * 10 ⁇ 10 M, determined in the experiments on entry of rhodamine- labeled peptide and peptide 182 toxicity tests on CHSE-214 cells.
- CHSE-214 cells at 0.8 ⁇ 10 4 cells / ml were seeded in two 24 well plates. 24 hours after sowing, the cells were treated for 4 hours with 200ul_ of peptide 182 in molar concentrations. As in the previous cases, the peptide was solubilized in L-15 medium without serum. After treatment with peptide 182, the cells were washed 2 times with 1 x PBS and subsequently infected with an MOI of 1 x IPNv which had a titer of 7.6 * 10 7 . Inoculation with the virus was carried out for 1 hour at 17 ° Celsius, followed by a gentle washing with 1 x PBS. Maintenance of the infected cells was performed with 1 ml of L-15 medium supplemented with 2% SFB at 17 ° C for 16 hours post infection (hpi). ( Figure 3)
- CHSE-214 cells were seeded and infected according the previously described protocols. Postinfection and after washing with 1 x PBS, cells were incubated with decreasing molar concentrations of peptide 182 from 10 ⁇ 4 to 10 ⁇ 10 M for 4 hours, the peptide was removed, the cells were washed with 1 x PBS and kept in 1 ml of L-15 medium with 2% SFB for 16 hpi ( Figure 4).
- the expression of the VP2 protein was measured by quantifying relative to elongation factor ELF-1 alpha of the CHSE-214 cells using RNA extraction and realtime PCR described in methodology of objective 2.
- the reduction in viral titre was evaluated using the procedure described in the literature and mentioned above, assessment in this case only was performed using a commercial monoclonal antibody against VP2 and with decreasing concentration of the peptide from 10 4 to 10 ⁇ 8 M.
- a unit was designed with 5 culture systems (experimental lines) with independent recirculating fresh water.
- Each culture system is composed of two culture units with a maximum capacity of 20 liters each, the system consists of a trickling 25L biofilter for the removal of solids and nitrogen compounds and a drain tank of 200L for the removal of residual water and feces.
- the water removed from the tanks post circulation is treated in the drain with ozone (0 3 ) and UV light to remove contaminants before returning to the sewer system, ensuring the removal of the remnants of the challenge virus.
- fry were separated into groups of 250 individuals per unit area, there were a total of 500 fry per culture system (experimental line).
- the culture conditions used were similar to those used in industrial fish farms, ensuring that these said conditions did not affect the development of individuals nor influence the results of the challenge.
- the general conditions correspond to a density of 25kg/m3 culture, with cycles of 16 hours light and 8 hours dark.
- Each tank was oxygenated in a ratio of 7 mg / L and the rate of water exchange in the system was 250 ml per minute, where the total turnover is within 4 hours.
- Table 1 Conditions of culture production used in the in vivo challenge in the wet lab at UCSC.
- the preliminary screening was performed using VP2SNP-F and VP2SNP-R primer set using the protocols for real-time PCR previously described in Methods.
- the experimental evaluation was conducted for 56 days, after the acclimatization of the fry.
- the application of peptide 182 was performed in a bath by reduction of the water column.
- the VR- 299 strain of IPNv obtained from cell cultures of CHSE-214 was used, as described previously in Methods.
- the virus was titered by IFAT at around 7.6 ⁇ 10 7 particles / ml.
- the water column was reduced, leaving the Salmo salarhy at a density of 50 kg/m3.
- Two types of treatment with the peptide described as pre- challenge treatment and post-challenge treatment according to the similar condition used in the in vitro model were performed.
- Table 2 Sample experimental design with peptide 182 (p182). Every situation has two separate tanks with 250 Salmo salar try, average weight 1.4 g.
- the initial state of the Salmo salar hy with respect to preexisting conditions was determined by an initial sampling which indicated that 60% of sampled fish were found to have IPN viral load. This is consistent with the literature which explains that the virus becomes persistent in fish that survive an outbreak and therefore the offspring may be asymptomatic carriers of the virus. Because of this, the untreated tank was considered to represent the natural mortality rate which may exist due to the activation of virus in the experimental tanks and the handling of the water column in which they were maintained. (Figure 9).
- the peptide p182 was applied by bath immersion to 300,000 fry with the company Marine Harvest. Samples were taken at 0, 30 and 60 days of application of the peptide and 25 specimens were analyzed.
- the fish were distributed in 5 tanks of 40 fish each per formulation (Table 5). Environmental parameters were measured in each tank (temperature in ° C and % oxygen saturation) daily for 45 days of the trial, which remained constant throughout the time.
- liver kidney and heart of each fish was sent to GIM-PUCV individually preserved in RNA later ®.
- SECOND STAGE developed at IMT-PUCV. To determine the viral load of fish analyzed, 180 untreated fish were taken at time "0" and the variation in Ct values was quantitatively determined in reference to the ratio of viral VP2 gene / cellular ELF. Four time points were analyzed in the experiment (days 10, 20, 30 and 45), removing 25 fish per formulation for which the ratio was Ct Vp2/ELF was determined in duplicate except for day 45 when 25 fish per formulation were tested without duplicates, in a pool of organs per fish of which 30 mg were taken to extract RNA with E.Z.N.A. kit for qRT-PCR.
- Table 8 Controls. Persistently infected fish, maximum and minimum values of Ct for the VP2 gene compared to the cell marker ELF.
- the present invention provides peptides that are suitable for pharmaceutical or veterinary compositions, which can help in the prophylaxis of viruses of the Birnaviridae family, more particularly, for the prophylaxis of fish that can be exposed to IPNv.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
Description
Claims
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/IB2014/063047 WO2016005796A1 (en) | 2014-07-11 | 2014-07-11 | Agent with antiviral properties for preventing or treating individuals exposed to a virus of the birnaviridae family |
Publications (2)
Publication Number | Publication Date |
---|---|
EP3166621A1 true EP3166621A1 (en) | 2017-05-17 |
EP3166621A4 EP3166621A4 (en) | 2018-05-09 |
Family
ID=55063642
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP14896988.4A Withdrawn EP3166621A4 (en) | 2014-07-11 | 2014-07-11 | Agent with antiviral properties for preventing or treating individuals exposed to a virus of the birnaviridae family |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP3166621A4 (en) |
AR (1) | AR101932A1 (en) |
AU (1) | AU2014400580A1 (en) |
CA (1) | CA2954876A1 (en) |
WO (1) | WO2016005796A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108129570B (en) * | 2017-12-25 | 2020-09-25 | 深圳市前海金卓生物技术有限公司 | Preparation and application of fusion bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU4970193A (en) * | 1992-08-26 | 1994-03-15 | Proteus Molecular Design Limited | IPNV vaccine |
WO2003013597A1 (en) * | 2001-08-10 | 2003-02-20 | University Of Maryland Biotechnology Institute | Sub-unit vaccine for infectious pancreatic necrosis virus |
GB2477712A (en) * | 2006-12-20 | 2011-08-17 | Advanced Bionutrition Corp | Antigenicity of infectious pancreatic necrosis virus VP2 sub-viral particles expressed in yeast |
WO2010111565A2 (en) * | 2009-03-27 | 2010-09-30 | Advanced Bionutrition Corporation | Microparticulated vaccines for the oral or nasal vaccination and boostering of animals including fish |
NO332608B1 (en) * | 2011-03-16 | 2012-11-19 | Norwegian School Of Veterinary Science | Live avirulent IPNV and vaccine for prophylaxis or treatment of IPN disease in fish |
WO2014052378A2 (en) * | 2012-09-26 | 2014-04-03 | Zoetis Canada Inc. | Subunit immersion vaccines for fish |
-
2014
- 2014-07-11 WO PCT/IB2014/063047 patent/WO2016005796A1/en active Application Filing
- 2014-07-11 AU AU2014400580A patent/AU2014400580A1/en not_active Abandoned
- 2014-07-11 CA CA2954876A patent/CA2954876A1/en not_active Abandoned
- 2014-07-11 EP EP14896988.4A patent/EP3166621A4/en not_active Withdrawn
-
2015
- 2015-07-13 AR ARP150102220A patent/AR101932A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
AU2014400580A1 (en) | 2017-02-16 |
WO2016005796A1 (en) | 2016-01-14 |
EP3166621A4 (en) | 2018-05-09 |
CA2954876A1 (en) | 2016-01-14 |
AR101932A1 (en) | 2017-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Essbauer et al. | Viruses of lower vertebrates | |
Lightner | Virus diseases of farmed shrimp in the Western Hemisphere (the Americas): a review | |
Lu et al. | The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model | |
Rimstad et al. | Infectious salmon anaemia | |
Kibenge et al. | Aquaculture virology | |
Zorriehzahra | Viral nervous necrosis disease | |
Qin et al. | First isolation and identification of red-grouper nervous necrosis virus (RGNNV) from adult hybrid Hulong grouper (Epinephelus fuscoguttatus× Epinephelus lanceolatus) in China | |
Gui et al. | Disease prevention and control | |
Godwin et al. | Pilchard orthomyxovirus (POMV). II. Causative agent of salmon orthomyxoviral necrosis, a new disease of farmed Atlantic salmon Salmo salar | |
Duan et al. | An inactivated vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss) | |
Garver et al. | Characterization of infectious hematopoietic necrosis virus (IHNV) | |
Mork et al. | Isolation and characterization of a rhabdovirus from starry flounder (Platichthys stellatus) collected from the northern portion of Puget Sound, Washington, USA | |
Gadd et al. | Characterization of perch rhabdovirus (PRV) in farmed grayling Thymallus thymallus | |
EP3166621A1 (en) | Agent with antiviral properties for preventing or treating individuals exposed to a virus of the birnaviridae family | |
Rud et al. | Experimental infection of brown trout (Salmo trutta), zebrafi sh (Danio rerio), and swan mussel (Anodonta cygnea) with infectious pancreatic necrosis virus (IPVN) | |
Munang’andu et al. | Birnaviruses of aquatic organisms | |
AU2014273193B2 (en) | Interactions of betanodaviruses in infection | |
Cowley | Nidoviruses of fish and crustaceans | |
JP5863839B2 (en) | Vaccine against Vibrio infection in shrimp and method for producing the same | |
Qayoom et al. | WSSV susceptibility in the early life stages of penaeus vannamei shows relationship with bodyweight | |
He et al. | Attenuation of a Highly Pathogenic Porcine Deltacoronavirus Strain CZ2020 by a Serial Passage In Vitro | |
Lio-Po | Recent developments in the study and surveillance of koi herpesvirus (KHV) in Asia | |
Nerland et al. | Viruses of fish | |
Matvienko et al. | Replication of Infectious Pancreatic Necrosis Virus in Different Cell Lines and in Rainbow Trout Fingerlings | |
Jin | Disinfection efficacy, mechanism and potential application of commercial proteases against fish viruses |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20170210 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: BA ME |
|
DAX | Request for extension of the european patent (deleted) | ||
A4 | Supplementary search report drawn up and despatched |
Effective date: 20180411 |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: A61K 38/16 20060101AFI20180405BHEP |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20181113 |