Recombinant eel herpesvirus 1 for preventing or treating diseases caused by eel herpesvirus 1

Abstract

The present invention relates to recombinant eel herpesvirus 1 for use in the prevention or treatment of diseases caused by eel herpesvirus 1. One aspect of the invention provides a live recombinant Eel Herpesvirus 1 (AngHV-1) comprising an open reading frame 35 (ORF35) dysfunctional genome. Further aspects provide products and compositions derived from or comprising live recombinant AngHV-1. Also provided are uses and methods for vaccination and disease treatment in fish, particularly eel, using live recombinant AngHV-1.

Description

Technical Field

[0001] This invention falls broadly in the field of veterinary medicine, and particularly relates to substances and compositions that can be used for the preventive and / or therapeutic treatment of diseases caused by eel herpesvirus 1 in fish, such as eels. Background Technology

[0002] Anguillid herpesvirus 1 (AngHV-1), also known as Cyvirusanguillidallo1, is an important eel virus belonging to the genus Cyprinivirus of the family Alloherpesviridae (now renamed Cyvirus). It poses a serious threat to both farmed and wild eel species worldwide. AngHV-1 is an enveloped double-stranded DNA virus that causes hemorrhagic skin lesions and gill destruction in various farmed and wild eel species, resulting in significant economic losses globally. For example, AngHV-1 has been frequently detected in European eels (Anguilla anguilla) from the Netherlands, Denmark, the United Kingdom, and Poland; Japanese eels (Anguilla japonica) from Japan and Taiwan; bicolor eels from South Korea; and more recently, marmorata eels from Vietnam and China (where eels are an important freshwater farmed fish).

[0003] The genus *Cyprinus* includes heteroherpesviruses of cyprinid fish (cyprinid herpesviruses 1, 2, and 3 (CyHV-1, CyHV-2, and CyHV-3)) and eels (eel herpesvirus 1 (AngHV-1)). CyHV-1 and CyHV-3 infect common carp and koi (Cyprinus carpio species), CyHV-2 infects goldfish, crucian carp, and silver carp (all of which are Carassius species), and AngHV-1 infects eels (Anguilla), including European eels and Japanese eels.

[0004] It is noteworthy that, unlike cyprinid fish, European eels and Japanese eels are spawning fish that migrate downstream. Their life cycle begins in the open ocean, with the larvae drifting with ocean currents to coastal waters of the continent, then migrating to inland freshwater habitats where they remain until they return to the ocean to spawn, after which they die. This complex life cycle, compared to other cypriniviruses, could have had a profound impact on the evolution of AngHV-1.

[0005] AngHV-1 has a mortality rate as high as 30% in fisheries, thus negatively impacting the industry. This virus is also considered one of the main reasons for the decline in the number of European eels, which are now listed as critically endangered (Bandín et al., Presence of Viruses in Wild Eels Anguilla Anguilla L., from the Albufera Lake (Spain). J. Fish Dis. 2014, Vol. 37, 597–60).

[0006] Clearly, a vaccine against AngHV-1 is highly anticipated. However, to date, there is no commercially available vaccine to combat eel disease caused by AngHV-1.

[0007] Boutier et al. (Rational development of an attenuated recombinant cyprinidherpesvirus 3 vaccine using prokaryotic mutagenesis and in vivobioluminescent imaging. PLoS Pathog. 2015, Vol. 11(2), e1004690) reported the development of a recombinant attenuated cyprinidherpesvirus 3 (CyHV-3) vaccine in carp based on a double-deleted recombinant CyHV-3 with deletions of ORF56 and ORF57. Boutier et al. noted the presence of a direct homolog of CyHV-3 ORF57 in AngHV-1, named it ORF35, and speculated that ORF35 might be a potential locus for generating attenuated recombinant vaccine candidates.

[0008] However, given the differences in epidemiological contexts between AngHV-1 and other Cypriniviruses (which infect very different host species with very different life cycles and behaviors (see above)), one would not expect too much similarity between AngHV-1 and the three Cyprinid herpesviruses CyHV-1, CyHV-2 and CyHV-3. It has been shown, in particular, that the conservation of structural genes of fish herpesviruses is limited for tegument proteins. For example, the AngHV-1 tegument protein ORF35 and CyHV-3 ORF57 share only 32% sequence identity (van Beurden et al., Identification and localization of the structural proteins of anguillidherpesvirus 1. Veterinary Research 2011, Vol. 42, 105; Van Beurden et al., Completegenome sequence and taxonomic position of anguillid herpesvirus 1. J GenVirol. 2010, vo 91, 880–887; Donohoe et al., Genomes of Anguillid Herpesvirus 1 Strains Reveal Evolutionary Disparities and Low Genetic Diversity in the Genus Cyprinivirus. Microorganisms. 2021, Vol. 9(5), 998).

[0009] Furthermore, recent research on the innate cellular defense mechanisms of European eel (Schulz et al., Immunosuppressive influence of Anguillid herpesvirus-1 (AngHV-1) infection on cellular defense mechanisms in European eel (Anguilla anguilla). Polish Journal of Veterinary Sciences 2019, Vol. 22(4), 785–787) showed that AngHV-1 induces a severe suppression of the phagocytic capacity and potential killing activity of splenic phagocytes compared to AngHV-1-negative eels. In addition, AngHV-1 reduces the proliferation of T lymphocytes and B lymphocytes isolated from the forearm of AngHV-1-positive eels compared to AngHV-1-negative eels. This would deter the use of live recombinant AngHV-1 as a vaccine base, as such a live vaccine could be expected to interfere with the host's immune system.

[0010] Therefore, it is understandable that current research on vaccines against AngHV-1 focuses on the development of subunit vaccines based on antigenic proteins. For example, Chen et al. (Anguillid herpesvirus 1 (AngHV) ORF95 encodes a late, structural envelope protein. Virus Genes 2021, Vol. 57, 280–283) identified ORF95 as a late virion envelope protein. ORF95 can play an important role in the AngHV-1 infection cycle, such as virion binding and mediating its entry, promoting virion morphogenesis, or facilitating virion expulsion from host cells. These results provide a new foundation for further elucidating the function of ORF95 in the AngHV-1 infection process and the possibility of using ORF95 as an antigen to develop AngHV-1 subunit vaccines.

[0011] Zhang et al. (Immune responses and protective efficacy of American eel (Anguilla rostrata) immunized with a formalin-inactivated vaccine against Anguillid herpesvirus. Fish and Shellfish Immunology 2024, Vol. 144, 109262) also pointed out the fact that no effective vaccine against AngHV-1 has been developed to date, and recently adopted the classic method of vaccination with a formalin-inactivated whole virus vaccine in 2024. However, vaccination with such an inactivated whole virus vaccine has an inherent drawback, in addition to the poor level of protection, namely that the vaccine must be administered to each fish individually by injection.

[0012] One object of the present invention is to provide a vaccine against AngHV-1. Summary of the Invention

[0013] The present invention is based, at least in part, on the inventors’ unexpected discovery of the highly anomalous behavior of live recombinant anguish herpesvirus 1 (AngHV-1) containing a dysfunctional open reading frame (ORF35).

[0014] In particular, the inventors have demonstrated for the first time that live recombinant AngHV-1 carrying a dysfunctional ORF35 (illustrated by ORF35 deletion) (AngHV-1 ΔORF35) has absolutely no effect on viral titers in cell cultures. This contrasts sharply with the live recombinant cyprinid herpesvirus 3 (CyHV-3) lacking both ORF56 and ORF57, or CyHV-3 lacking only functional ORF57, reported by Boutier et al. (Identification of an essential virulence gene of cyprinid herpesvirus 3. Antiviral Res. 2017, Vol. 145, 60-69), whose replication in cell cultures was significantly reduced in both cases compared to wild-type viruses. Given that this reduced replication in cell cultures is the basis for the attenuated phenotypes of live recombinant CyHV-3 ΔORF56 ΔORF57 and CyHV-3 ΔORF57 in fish, the inventors’ cell culture data suggest that AngHV-1 ΔORF35 is unlikely to be attenuated in fish and is therefore unsuitable as a vaccine.

[0015] Despite this initial setback, the inventors persisted with their experiments and discovered that, contrary to the expectation of undiminished replication of AngHV-1 ΔORF35 in cell cultures (i.e., the modified virus being as virulent as wild-type AngHV-1 in the fish host), administration of AngHV-1 ΔORF35 unexpectedly resulted in abortive, non-replicative infection in fish. Intraperitoneal injection of the ORF35-deficient recombinant virus, immersion in water containing the recombinant virus, and intradermal inoculation all resulted in abortive, non-replicative infection. Abortive viral infection is generally defined as an infection in which cells or the entire organism are infected by a virus but fail to produce progeny virions due to the infection. Therefore, abortive infection is a viral infection of cells or even the entire organism to the extent that the infecting virus cannot replicate to the extent that the infected cell or organism expresses signs of infection and does not release progeny virions. This contrasts with the results obtained from the deletion of the ortholog ORF57 in CyHV-2, which showed only a slight reduction in mortality induced in goldfish, thus identifying ORF57 as a non-essential virulence factor in CyHV-2 (Feng et al., Generation and Characterization of ORF55 / ORF57-Deleted Recombinant Cyprinid herpesvirus 2 Mutants with ChimericCapsid Protein Gene of Grouper Nervous Necrosis Virus. Vaccines 2023, Vol. 12, 43).

[0016] The occurrence of abortive infections would predict that AngHV-1 ΔORF35 would not induce any immunization, as the viral load generated in the fish (if any) would be insufficient to trigger an immune response. However, undeterred by these advances, the inventors continued to test the vaccination potential of AngHV-1 ΔORF35 in fish and, quite unexpectedly, discovered that administration of live recombinant AngHV-1 ΔORF35 conferred protection against wild-type AngHV-1 virus attack in the fish. This immune protection depends on the virus being live (infectious), as inactivating the same amount of virus through psoralen and UV treatment does not produce any protection.

[0017] From a vaccination safety perspective, this abortive characteristic of the virus is a significant advantage because although the vaccine virus clearly infects the host during vaccination, the host does not release new viral particles and therefore will not release the vaccine virus into the environment. Consequently, the vaccine virus cannot infect other fish, which is an advantage given the stringent regulations governing the release of genetically modified viruses into the environment. Furthermore, AngHV-1 ΔORF35 is effective even when fish are vaccinated by immersion in virus-containing water, a highly desirable route of administration from an economic standpoint. Therefore, despite these challenges, the inventors have determined that the ORF35-deficient live recombinant AngHV-1 virus is a highly desirable and safe vaccine agent against AngHV-1.

[0018] Therefore, one aspect of the present invention provides live recombinant eel herpesvirus 1 (AngHV-1) comprising an open reading frame 35 (ORF35) dysfunctional genome.

[0019] Other aspects include: nucleic acid molecules containing the live recombinant AngHV-1 genome; cells containing live recombinant AngHV-1 or containing nucleic acids; and pharmaceutical compositions containing live recombinant AngHV-1, nucleic acids and / or cells, and further comprising a pharmaceutically acceptable carrier and / or adjuvant.

[0020] On the other hand, live recombinant AngHV-1, nucleic acid, cells, or pharmaceutical compositions are provided for use in fish immunization. Live recombinant AngHV-1, nucleic acid, cells, or pharmaceutical compositions are also provided for the treatment of diseases in fish caused by herpesviruses.

[0021] On the other hand, a method for generating infectious particles of recombinant live AngHV-1 is provided, the method comprising the steps of: (a) introducing live recombinant AngHV-1 or a nucleic acid molecule as taught herein into a permitted eukaryotic cell; and (b) culturing the cell to produce live recombinant AngHV-1.

[0022] These and other aspects of the invention, as well as preferred embodiments, are described in the following sections and the appended claims. The subject matter of the appended claims is specifically incorporated herein by reference. Attached Figure Description

[0023] The following description of the figures of specific embodiments of the present invention is merely exemplary in nature and is not intended to limit the teachings, their application or use.

[0024] Figure 1. Schematic diagram of a strategy for generating AngHV-1 recombinants via homologous directed recombination (HDR). Figure 1AA flowchart illustrating the generation of recombinant UK Luc and ORF35 Del strains in eukaryotic cells via HDR. Figure 1B The UK parental strain and derived recombinant strains target the genotypes of the ORF32-ORF33 intergenic region and the ORF35 locus. WT, wild type; Luc, inserted LucGFP box; Del, deletion. Figure 1C A schematic diagram of the UK Luc genome structure. The two terminal repeat sequences (LTR and RTR) flanking the AngHV-1 genome and the intergenic ORF32 to ORF33 genomic regions are shown at the top. Figure 1D Schematic diagram of the genome structure of the UK ORF35 Del recombinant. The two terminal repeat sequences (LTR and RTR) flanking the AngHV-1 genome and the ORF35 genomic region are shown at the top. In Figures C and D, the SacI restriction sites and predicted restriction fragments (in kb) are shown. The coordinates are those of the AngHV-1 reference strain available in GenBank (accession number MW580855.1).

[0025] Figure 2 Characterization of recombinant AngHV-1 strains. Figure 2 A) Transcriptional analysis of genes ORF32, ORF33, ORF34, ORF35, ORF36, and ORF55 expressed by the specified AngHV-1 strain. ORF55 (AngHV-1 DNA polymerase) expression was used as a control. Marker size (MS) in base pairs (bp) is shown on the left. The left and right parts of the figure represent the results of PCR for cDNA and RNA, respectively. Figure 2B The expression of reporter genes was assessed. EK-1 cells grown in 12-well plates were infected with the specified strain and then covered with medium containing carboxyl methyl cellulose (CMC). Bioluminescence and epifluorescence expression in infected cells were analyzed at 4 dpi. Luc signal was detected using an IVIS system (left box). Reporter genes (copGFP and mCherry) and immunofluorescence staining (anti-AngHV-1) were detected by epifluorescence microscopy. Plaques of UK and UK ORF35 Del were shown by indirect immunofluorescence staining (anti-AngHV-1) (right box). Figure 2 C) Replication dynamics, and ( Figure 2 D) Compare the viral plaque size with that of the parental UK strain. Figure 2E) Luc expression in recombinant AngHV-1 strains (UK Luc and UK Luc ORF35 Del). Replication kinetic data are presented as mean ± SEM of triplicate measurements. Plaque area data are presented as mean ± SEM of twenty measurements. Luc expression data are presented as mean ± SEM of triplicate measurements. The horizontal dashed line in Figure E indicates the mean ± 3 SD of data obtained from uninfected control cultures. The time points at which significant differences were observed between the UK ORF35 Del strain and the UK parent strain are shown below: ns, no significant difference; * , p<0.05.

[0026] Figure 3. Effect of ORF35 deletion on AngHV-1 replication in vivo. Figure 3A Experimental flowchart. At inoculation, yellow eels (12.06 ± 2.72 g, mean ± SD) were either infected using a simulated infection method or infected with the specified strain via different routes: IP injection of 200,000 pfu / eel, immersion in water containing 4000 pfu / mL, or intradermal inoculation of 200,000 pfu / eel. At a specified time post-infection, the eels (n=6, consisting of two eels from triplicate tanks) were imaged using IVIS. Figure 3B and 3B continued The effects of AngHV-1 infection routes: IP injection, immersion, and intradermal inoculation are shown in the left, middle, and right columns, respectively. Left Y-axis: mean radiation - Log 10 [p / s / cm 2 / sr]; Right Y-axis: Number of positive eels (n = 6); X-axis: Number of days after infection. Mean radioactivity (single value, mean ± SEM) was measured on the entire body surface of the fish (single value representing the average obtained from the left and right sides of each fish), gills (single value representing the average obtained from the left and right gills), brain, heart, and intestine-liver (n=6 at each time point) by IVIS analysis. Dashed lines represent the positive threshold, which is the mean of values ​​obtained from simulated infected fish + 3 SD (data not presented). The number of positive fish out of six analyzed fish is represented by bars (right Y-axis). Mean radioactivity (p(rad)) of UK Luc was compared with that of UK Luc ORF35 Del using an unpaired t-test (two-tailed, Gaussian distribution) or the Mann Whitney test (non-Gaussian distribution). The number of positive fish per group was compared between the two strains using the Fisher-Pitman permutation test (p(no)). p-values ​​are expressed as follows, ns: not significant, * p<0.05, ** p<0.01, *** p<0.001.

[0027] Figure 4. Dose-protective effect of UK ORF35 Del in vivo. Figure 4A Experimental flowchart. At the initial inoculation, yellow eels (28.52 ± 7.30 g, mean ± SD) were either infected by simulating infection or by immersion in water containing a specified dose of UKORF35 Del or 100,000 pfu / mL of UK ORF35 Del strain inactivated by psoralen / UV treatment for 2 hours. Thirty-six days after the initial inoculation, eels were infected by immersion in water containing 4,000 pfu / mL of UK Luc expressing luciferase as a reporter gene for 2 hours. At a specified time after the second inoculation, eels (n=6, consisting of two eels from triplicate tanks) were imaged using IVIS. Figure 4B and 4B continued Mean radioactivity (single value, mean ± SEM) was measured on the entire body surface of the fish, i.e., skin (single value represents the average obtained from the left and right sides of each fish), gills (single value represents the average obtained from the left and right gills), brain, heart, and intestine-liver (n=6 at each time point) using IVIS analysis. The mean radioactivity (p(rad)) of each group was compared to the “initial simulated infection group” using the nonparametric Kruskal-Wallis test, followed by multiple comparisons using the two-stage incremental method of Benjamini, Krieger, and Yekutieli. Throughout the figure, data obtained from each individual eel (within each group) are represented by the same symbol to allow for correlation of data obtained from different organs at a specific dpi. Dashed lines represent the positive threshold, calculated as the mean of values ​​obtained from simulated fish + 3 × SD. The number of positive fish among the six analyzed fish is represented by a bar (right Y-axis). The positive fish (p(no)) from each group were compared to the initial simulated infection group using the Fisher-Pitman permutation test. p-values ​​are expressed as follows, ns: not significant. * p<0.05, ** p<0.01. Detailed Implementation

[0028] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein include both the singular and plural referents.

[0029] As used herein, the term “comprising” is synonymous with “including” or “containing” and is inclusive or open-ended, without excluding additional undescribed members, elements, or method steps. The term also covers “constituting of,” “existing in,” and “component of,” as well as the terms “substantially constitutes of” and “substantially exists,” which have their generally accepted meanings in patent terminology.

[0030] The description of a numerical range by endpoints includes all intermediate values ​​between the lower and upper endpoints, as well as the recorded endpoints. Intermediate values ​​can be integers, or, where applicable, fractions, i.e., more broadly any real number, such as any rational number. This applies to numerical ranges regardless of whether they are introduced by the expression "from...to...", "between...and...", or another expression. Any numerical range described herein is intended to include all subranges contained therein. For example, each subrange between any stated value within the range and any other stated value within the range is also specifically disclosed. Each subrange between any stated value within the range and the lower or upper endpoint of the range is also specifically disclosed. The value can be an isolated value or an endpoint of a range that is contained within or overlaps with the range. For example, for the range having a lower endpoint L1 and an upper endpoint U1 (i.e., the range L1-U1) and the sub-range nested within the range having a lower endpoint L2 and an upper endpoint U2 (i.e., the sub-range L2-U2), sub-ranges L1-L2, L1-U2, L2-U1 and U2-U1 are also specifically disclosed.

[0031] When referring to measurable values ​​such as parameters, quantities, and temporal durations, the term "about" or "approximately" as used herein means encompassing variations in a specified value and variations relative to that specified value, such as + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and even more preferably + / -0.1% or less, and variations relative to a specified value of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and even more preferably + / -0.1% or less, within which such variations are suitable for implementation in the disclosed invention. It should be understood that the values ​​referred to by the modifier "about" or "approximately" are themselves specifically and preferably disclosed.

[0032] Furthermore, the terms first, second, third, etc., used in the specification and claims are used to distinguish between similar elements and are not necessarily used to describe a sequential or temporal order, unless specifically stated otherwise. It should be understood that such terms are interchangeable where appropriate, and embodiments of the invention described herein can operate in orders other than those described or illustrated herein.

[0033] While the terms "one or more" or "at least one" (e.g., one or more members of a group of members or at least one member) are self-evident, by further example, the term specifically covers any one of the members, or any two or more of the members, such as any ≥3, ≥4, ≥5, ≥6, or ≥7 members, and at most all of the members mentioned. In another instance, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0034] As used herein, the term “and / or” when used in a list of two or more items means that any one of the listed items may be used alone, or any combination of two or more of the listed items may be used. For example, if a list is described as containing groups A, B, and / or C, then the list may contain: A alone; B alone; C alone; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0035] This includes a discussion of the background of the invention herein to explain the context of the invention. This should not be construed as an admission that any material mentioned has been disclosed, known, or is part of general common knowledge in any country since the priority date of any claim.

[0036] Throughout this disclosure, numerous publications, patents, and published patent specifications are cited by way of reference. All documents cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings or portions of such documents specifically mentioned herein are incorporated by reference.

[0037] Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance, including terminology definitions, is provided to better understand the teachings of this invention. Unless otherwise defined, when a particular term is defined in connection with a specific aspect or embodiment of the invention, such connotation or meaning is intended to apply throughout this specification, that is, also in the context of other aspects or embodiments of the invention.

[0038] The following paragraphs define different aspects or embodiments of the invention in more detail. Each aspect or embodiment so defined may be combined with any other aspect or embodiment unless explicitly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with one or more other features indicated as preferred or advantageous.

[0039] Throughout this specification, references to "an embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, as will be apparent to those skilled in the art based on this disclosure, particular features, structures, or characteristics may be combined in any suitable manner. Moreover, while some embodiments described herein include certain features but not others included in other embodiments, as will be understood by those skilled in the art, combinations of features from different embodiments are intended to be within the scope of the invention and form different embodiments. For example, in the appended claims, any claimed embodiment may be used in any combination.

[0040] Similarly, it should be recognized that in the description of exemplary embodiments of the invention, various features of the invention are sometimes combined in a single embodiment, figure or description thereof for the purpose of simplifying the disclosure and aiding in understanding one or more aspects of the invention.

[0041] As demonstrated by the experimental portion illustrating certain representative embodiments of the invention, the inventors have shown that the ORF35-deficient live recombinant AngHV-1 virus is an unexpected, highly advantageous, and safe vaccine against AngHV-1.

[0042] One aspect of the invention relates to live recombinant eel herpesvirus 1 (AngHV-1), which contains a dysfunctional open reading frame 35 (ORF35) genome. Eel herpesvirus 1 (AngHV-1), also known as Cyvirusanguillidallo 1, is a virus belonging to the genus Cyvirus (now renamed Cyvirus), family Heteroherpesviridae, and order Herpesvirales. This virus is known to infect eels, including, but not limited to, the Japanese eel (A. anguilla), the European eel (A. rostrata), the Indonesian shortfin eel (A. bicolor), and the giant mottled eel (A. marmorata). Sano et al. (Isolation and Characterization of a New Herpesvirus From Eel. Pathology in Marine Science, Academic Press, 1990, pp. 15–31) and Davidse et al. (First isolation of herpesvirus of eel (Herpesvirus anguillae) indiseased European eel (Anguilla anguilla L.) in Europe. Bulletin of the European Association of Fish Pathologists. 1999, Vol. 19 (4), pp. 137–141) have isolated the virus.

[0043] AngHV-1 is an enveloped double-stranded DNA virus, and its wild-type genome contains more than 130 open reading frames (ORFs) encoding proteins, such as the 133 ORFs in the isolate published by Kole et al., Complete Genome Sequence of Anguillid Herpesvirus 1 Isolated from Imported Anguilla rostrata (American Eel) from Canada. Microbiol Resour Announc. 2022, Vol. 11(12): e00829-22. The complete genome sequences of several illustrative non-restrictive isolates of AngHV-1 have previously been obtained from the National Center for Biotechnology Information (NCBI) Genbank (http: / / www.ncbi.nlm.nih.gov / ) of the U.S. government, accession numbers OM936983.1 (AngHV-1 isolate 16023, complete genome; see also Kole et al., ibid.), KX027736.1 (AngHV-1 isolate HVA980811, complete genome), FJ940765.3 (AngHV-1 strain 500138, complete genome), NC_013668.3 (AngHV-1, complete genome) and OM649903.1 (AngHV-1 strain FC2021, complete genome). Genbank accession number MW580855.1 provides a partial genome sequence of the anchovy herpesvirus 1 strain UK N080. Isolates of AngHV-1 show low genetic diversity but exhibit a range of eel host species and a wide geographic distribution (Kole et al., ibid.).

[0044] AngHV-1 strains or isolates can be easily isolated from tissues collected from infected eels (such as kidneys, spleens, livers, or hearts) or from swabs taken from the gills or mouth. In one exemplary technique, tissue is homogenized in a sterile buffer solution (e.g., phosphate-buffered saline, PBS), the homogenate is centrifuged to remove debris, and the supernatant is typically seeded onto an approved cell line, such as EK-1 (eel kidney) cells (Chen et al., A cell line derived from Japanese eel (Anguilla japonica) kidney. Proc. Natl. Sci. Counc. Repub. China, B, Life Sci. 1982, Vol. 6, 93-100; disclosed from IZSLER Biobank of Veterinary Resources, Via Antonio Bianchi 7, 25124 Brescia, Italy, accession code BS CL 232, which can be cultured in Earle's BSS + 10% fetal bovine serum on Eagle's minimally essential medium, for example) or EO (eel ovary) cells or EB (eel brain) cells (e.g., Bloch et al., Development of a cell line). From the American eel brain expressing endothelial cell properties. In Vitro Cell Dev Biol Anim. 2016, Vol. 52(4), 395-409. Further guidance on the development and culture of fish cell lines can be found, for example, Lakra et al., Development, characterization, conservation and storage of fish cell lines: a review. FishPhysiol Biochem. 2011, Vol. 37(1), 1-20 or Kumar et al., Fish cell line: depositories, web resources and future applications. Cytotechnology 2024, Vol. 76(1), 1-25. Incubate the cells at a suitable temperature, typically around 20 to 25°C, which is optimal for both cell and AngHV-1 replication.Herpesvirus cytopathic effects, i.e., changes in cell morphology caused by viral infection, such as cell rounding, cell enlargement, cell detachment, and / or syncytial formation, can be observed in cell cultures. The presence of AngHV-1 is confirmed using appropriate assays, such as, but not limited to, PCR (polymerase chain reaction, e.g., using primer pairs targeting the AngHV-1 DNA polymerase gene) for detecting viral DNA, immunofluorescence or ELISA (enzyme-linked immunosorbent assay) using antibodies specific to AngHV-1, or electron microscopy for visualizing viral particles. The virus can be further multiplied by infecting fresh cultures with the supernatant of infected cells, and the virus can be harvested by collecting the culture supernatant when the maximum cytopathic effect is observed, followed by further purification if desired. Isolated viruses can be stored at -80°C or in liquid nitrogen for long-term preservation.

[0045] Open reading frame 35 (ORF35) exhibits high conservation among AngHV-1 isolates from different eel species. For example, Kole et al. (ibid.) reported 100% sequence identity in BLASTN sequence alignment among the nucleic acid sequences of ORF35 from American eel (Genbank accession number OM936983.1), Japanese eel (A. japonica) (Genbank accession number KX027736.1), and European eel (Genbank accession number FJ940765.3).

[0046] To illustrate, the protein sequence encoded by ORF35, annotated with Genbank accession number OM936983.1, is reproduced from the N-terminus to the C-terminus as follows (SEQ ID NO: 1):

[0047] MNGELSVDHAKLIGKKSPFGCYGPFEVAKFLVAVPERKWTEEHTNMMNAALGVEAHLHATVSHKIFVVGTAQLFQQLWHGIKRMPDMINSVFDHDASKLTLLERIVYGLMMSVQESLTLSEAGQPVVSAFTPDLVKEMYRWIVQKGFN HHYVLNQHHPQHWGMLPMSEAAVREAVIDGLAVVLERVPTITTVEDLLSRYRIPKNHNEPMFRAVLNDFRRAAGNNVDLPAVRKLLTDLVKLGFINLLPAWSSLGVPPPSNPPIFTFGDCLCDSMVANKTLPMCKGDYYHDTGKVPPQ

[0048] By way of example, the nucleic acid sequence of ORF35 annotated with Genbank accession number OM936983.1, from position 55585 (nucleotides 55585 to 55587 correspond to the start codon) to position 56475 (nucleotides 56473 to 56475 correspond to the stop codon) of the OM936983.1 genome sequence, is reproduced from the 5' to 3' end as follows (SEQ ID NO: 2):

[0049] atgaacggcgaactttccgtggatcacgccaaactcatagggaagaagtctcccttcgggtgctacggacctttcgaggtcgccaaatttttggtcgcggtacccgagaggaaatggacagaggaacacacgaacatgatgaacgcagcgcttggggtggaggctcacctgcacgccaccgtctctcacaagattttcgtcgtggggacggcgcagctgtttcaacaactgtggcacggaatcaaacgtatgcccgatatgataaacagcgtgttcgatcacgacgcctcaaaactgactctgttggaacgcatcgtctacggattgatgatgagcgttcaagagagtctgactctcagcgaagccgggcagcccgtcgtgtcggctttcacacccgacttggtgaaagagatgtaccggtggatcgtgcaaaagggtttcaatcaccactacgtgctgaaccagcatcacccgcaacactggggcatgctaccaatgtcagaagctgcggtgagagaggcggtgattgacggattggcagtggttctggagagggtccccaccatcaccacggtggaagaccttctgtcccgttacaggattccgaagaatcataacgaacccatgttcagagccgtgctgaacgatttccgcagagcggccgggaacaacgtggatctacccgctgtgaggaagttgctcaccgatttggtgaaactgggtttcatcaaccttctgcccgcatggtcttctctcggagtcccaccaccgtccaaccctcctatcttcactttcggagactgtctctgcgactctatggtcgccaataaaactctacccatgtgtaaaggggattactaccacgacactggcaaagttcctccccagtag

[0050] A skilled technician can identify ORF35 in the genome of any AngHV-1 isolate or strain, for example, based on a sufficient degree of sequence identity between the ORF35 nucleic acid or amino acid sequence in that isolate or strain and the aforementioned nucleic acid or amino acid sequence of ORF35 in Genbank accession number OM936983.1. A skilled technician understands that, due to normal genetic diversity (polymorphism) within viral species, the ORF35 nucleic acid or amino acid sequence can differ between different AngHV-1 isolates or strains. A skilled technician also understands that ORF35 can be located at different nucleotide positions in different AngHV-1 isolates or strains, i.e., at positions other than positions 55585 to 56475 in Genbank accession number OM936983.1. Therefore, the reference to AngHV-1 open reading frame 35 (ORF35) as used herein covers any AngHV-1 open reading frame having at least 80% sequence identity, such as having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the DNA sequence of ORF57 given in Genbank accession number OM936983.1 in ascending order of priority.

[0051] In the context of nucleotide sequences, the terms “identity,” “sequence identity,” or “identical” are used interchangeably herein and refer to the degree to which nucleic acid sequences are identical on a nucleotide-by-nucleotide basis. The percentage of sequence identity can be calculated by comparing two best-aligned sequences, determining the number of positions in both sequences where the same nucleic acid base occurs to generate the number of matching positions, dividing the number of matching positions by the total number of positions, and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence identity between nucleic acids can be conveniently determined using suitable algorithms known per se for sequence alignment and sequence identity determination. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2 Sequences” tool described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), or the “blastn suite-2sequences” sequence alignment algorithm described by Zheng Zhang et al. 2000 (J Comput Biol 2000, Vol. 7(1-2), 203-14), which is now incorporated into the BLAST program suite and is available at ncbi.nlm.nih.gov. Those skilled in the art can implement such algorithms and set the necessary parameters. By way of example rather than restriction, the parameters of the BLASTN program can be as follows: gap opening penalty = 0, gap extension penalty = 2.5, matching reward = 1, mismatch penalty = -2, expected value = 0.05, word length = 28, low complexity filter = yes.

[0052] As used herein, a “disabled” or “deficient” ORF, such as, in particular, ORF35, is an ORF whose function has been reduced or eliminated compared to the function of a wild-type ORF. Specifically, a disabled or deficient ORF can lead to reduced or eliminated expression or activity of the protein encoded by the ORF. In some preferred embodiments, a disabled or deficient ORF can lead to reduced or eliminated expression of the protein encoded by the ORF. The term “expression” of a protein by a cell generally refers to the production or synthesis of said protein by the cell. The term “activity” of a protein as used herein should be interpreted broadly and can generally encompass any one or more aspects of the biological activity of a protein at any level (e.g., molecular, cellular, and / or physiological), such as, but not limited to, any one or more aspects of its biochemical activity, enzymatic activity, signal transduction activity, interaction activity, ligand activity, receptor activity, or structural activity. By way of example, but not limited to, a dysfunctional ORF may still encode the corresponding protein, i.e., contain the complete coding sequence of the corresponding protein, but compared with a wild-type ORF, the expression of the protein may be reduced or eliminated, for example, because one or more regulatory sequences (such as transcription promoters or enhancers that control ORF transcription) have been conferred defectively. By way of another example, but not limited to, a dysfunctional ORF may no longer encode a functional protein; for example, the ORF may be completely absent, or the ORF may have been mutated, thereby encoding a mutant variant of the corresponding protein, such as a truncated variant of the corresponding protein, or a protein variant containing one or more amino acid substitutions, deletions, or insertions, thereby reducing or eliminating the expression or activity of the protein.

[0053] The term “reduction” is intended to be synonymous with terms such as “inhibition,” “reduction,” or “weakening,” and the term “elimination” is intended to be synonymous with terms such as “elimination” or “eradication.” These terms respectively indicate a partial or complete qualitative or quantitative decrease in the function of the ORF or in the expression or activity of a protein encoded by the ORF (e.g., ORF35). Any degree of reduction in the expression or activity of a protein encoded by ORF35 is contemplated, which achieves the objective of the invention, namely, the production of live recombinant AngHV-1 for immunization or treatment of fish, particularly in which live recombinant AngHV-1 induces abortive infection in eels. By way of illustration, a reduction in the expression or activity of a protein encoded by an ORF (e.g., ORF35) is considered to be at least 50% lower than the expression or activity of a protein encoded by the corresponding wild-type ORF, for example, at least 60%, at least 70%, at least 80%, or at least 90% or more in an increasing order of priority. Technicians are familiar with standard methods for measuring the amount of nucleic acids (e.g., mRNA) or proteins, such as determining the expression level of ORFs, or standard methods for measuring protein activity (e.g., quantitative reverse transcription PCR (qRT-PCR), immunoassays such as Western blot or ELISA, etc.).

[0054] ORFs, such as ORF35 in particular, can be dysfunctional or defective, for example, by introducing mutations affecting genes containing said ORF and / or their regulatory sequences into the genome of wild-type AngHV-1. Any mutations that result in dysfunctional or defective ORFs are considered herein, such as, in particular, a reduction or elimination of the expression or activity of the protein encoded by the ORF. Suitable mutations, for example, may include deletions, insertions, and / or substitutions. The term “deletion” refers to a mutation in which one or more nucleotides (typically consecutive nucleotides) of a nucleic acid are removed from the nucleic acid. The term “insertion” refers to a mutation in which one or more nucleotides (typically consecutive nucleotides) are added to the nucleic acid. The term “substitution” refers to a mutation in which one or more nucleotides of a nucleic acid are each independently replaced by another nucleotide.

[0055] In some implementations, mutations can introduce an in-frame early stop codon into the ORF. Such an early stop codon can result in the production of a C-terminal truncated form of the corresponding protein. This can preferably affect, for example, reduce or eliminate some or all of the protein's biological function, or, particularly when the stop codon is introduced near the ORF translation start codon (e.g., downstream of about 20 or fewer, or about 10 or fewer amino acids), the stop codon can effectively eliminate protein production. Various methods for introducing an in-frame early stop codon will be apparent to those skilled in the art. For example, but not limited to, a suitable insertion, deletion, or substitution of one or more nucleotides in the ORF can introduce an in-frame early stop codon.

[0056] In some implementations, the mutation may introduce a frameshift (e.g., +1 or +2 frameshift) into the ORF. Typically, such a frameshift causes a previous out-of-frame stop codon downstream of the mutation to become an in-frame stop codon. Therefore, such a frameshift can result in the production of a protein form with an alternative C-terminal portion and / or a C-terminal truncated form of said protein. This can preferably affect, for example, reduce or eliminate some or all of the protein's biological function, or, particularly when the stop codon is introduced near the ORF translation start codon (e.g., about 20 or fewer, or about 10 or fewer amino acids downstream), the stop codon can effectively eliminate protein production. The various ways in which a frameshift can be introduced will be apparent to those skilled in the art. For example, but not limited to, the appropriate insertion or deletion of one or more nucleotides (not multiples of 3) in the ORF can result in a frameshift.

[0057] In other embodiments, the mutation may delete at least a portion of the ORF. Such deletion may result in the production of an N-terminal truncated form, a C-terminal truncated form, and / or an internal deletion form of the protein. This may preferably affect, for example, reduce or eliminate some or all of the protein's biological function. Preferably, the deletion may remove about 20% or more, or about 50% or more, of the nucleotides in the ORF. In particular, when the deletion removes a substantial portion of the ORF (e.g., about 50% or more, preferably about 60% or more, more preferably about 70% or more, even more preferably about 80% or more, and still more preferably about 90% or more), or when the deletion removes the entire ORF, the deletion can effectively eliminate the production of the protein. Such deletions can be readily introduced by those skilled in the art.

[0058] In other implementations, the mutation may omit at least part or all of the regulatory sequence that controls ORF transcription, such as part or all of the promoter, resulting in impaired transcription of the gene product.

[0059] In some alternative embodiments, the mutation may be a substitution of one or more nucleotides in the ORF that results in the substitution of one or more amino acids in the protein. Such mutations generally preserve protein production and may preferably affect, for example, reduce or eliminate some or all of the protein's biological functions. Such substitutions can be readily introduced by those skilled in the art.

[0060] Therefore, in some embodiments, a dysfunctional or defective ORF, such as, in particular, ORF35, contains the insertion, deletion, and / or substitution of one or more nucleotides in the gene containing ORF35 or in its promoter region. Exemplary mutations include frameshift mutations adjacent to the 5' end of the ORF, or deletions of a portion or the entire promoter region, or deletions of a portion or the entire gene itself. From a practical and safety (i.e., mutation stability) perspective, a complete deletion of an ORF (e.g., ORF35), i.e., a deletion produced in the genome where the deletion covers the entire ORF, may be preferred. Preferably, the deletion should not affect the function of adjacent genes (including their regulatory sequences and the ORF). Therefore, in some embodiments, live recombinant AngHV-1 is provided, wherein ORF35 is completely or partially deleted.

[0061] AngHV-1 can be genetically manipulated using techniques available in the art, such as homologous directed recombination (HDR) in suitable eukaryotic cells using a parental AngHV-1 strain, as illustrated in the examples. In this technique, a genetic construct comprising nucleic acids containing the desired genetic alterations to be introduced into the parental AngHV-1 strain, such as mutations that would impair or defect ORF35, is prepared, flanked by two homologous arms corresponding to AngHV-1 sequences present upstream and downstream of the target locus. These homologous arms can typically be several hundred base pairs long, for example, typically about 500 bp long. The nucleic acid construct is transfected into an AngHV-1-enabled cell line, such as the EK-1 cell line, and the cells are infected with the parental AngHV-1 strain at a predetermined time after transfection, such as the day after transfection. Subsequently, progeny recombinant AngHV-1 strains that have incorporated the genetic alterations are selected, for example, by genetic sequence analysis. Optionally, genes encoding selection markers may be included in the construct to facilitate the selection process.

[0062] The term "recombinant" is generally used to indicate that materials (e.g., nucleic acids, genetic constructs, proteins) have been altered through technological means (i.e., non-naturally) by human intervention. This term is commonly used to describe nucleic acids, particularly DNA molecules, or viruses, cells, or organisms containing such nucleic acids, where the nucleic acids have been artificially synthesized or modified in vitro or in a suitable biological system to produce sequences not originally present in the genome. Techniques that can be used to produce recombinant nucleic acids include, but are not limited to, nucleic acid synthesis, nucleic acid amplification, molecular cloning, homologous directed recombination, and gene editing techniques known in the art. Therefore, recombinant AngHV-1 refers to AngHV-1 containing a genome that has been modified or altered using recombinant DNA technology compared to wild-type AngHV-1.

[0063] As taught herein, the recombinant AngHV-1 is a live virus. The term "live virus" refers to a virus that retains the ability to replicate in a cell culture, rather than a virus that has been inactivated or killed to render it non-infectious through chemical, physical, or radioactive processes. The viability of the recombinant AngHV-1 virus of the present invention can be tested, for example, by demonstrating infection and replication of the virus in a susceptible cell culture system, such as in eel EK-1 cells.

[0064] Live recombinant AngHV-1 preferentially induces abortive infection in eels. Abortive infection occurs when a virus enters a host organism but fails to replicate effectively, resulting in very limited or no progeny virions. This type of infection is characterized by the virus's inability to achieve productive infection by releasing new virions capable of infecting other cells. Therefore, the virus has the ability to infect fish, particularly eels, but cannot replicate to the extent necessary to form infectious progeny viruses and cause symptoms. The presence of abortive infection can be detected, for example, by infecting one experimental eel or a statistically significant number of experimental eels with recombinant live AngHV-1 containing a dysfunctional ORF35, and infecting a control eel of the same species or a statistically significant number of control eels with the same number of other similar but functional live AngHV-1 particles containing a functional ORF35 (e.g., wild-type AngHV-1), incubating the experimental and control eels under identical conditions, and comparing the amount of new virions produced in selected tissues of the experimental and control eels at a selected time after the initial infection, or by comparing signals associated with reporter proteins (e.g., luciferase) that are comparablely expressed by the two viruses. Different methods can be used to compare the replication levels of the two viruses. Viral load can be quantified by the quantification of viral DNA or one or more viral proteins, or the number of virions can be determined by infecting susceptible cell culture systems using limiting dilution, or, in the case of comparing wild-type and ORF35-deficient strains that comparablely express reporter proteins, by quantifying the expression of reporter proteins. For example, abortive infection can be inferred when the following occurs: the amount of progeny virions produced from live recombinant AngHV-1 containing a dysfunctional ORF35, or the expression of the reporter gene (in the case of a comparison between wild-type and ORF35-deficient strains expressing the reporter protein), at at least one measurement time after initial infection, and preferably at all selected measurement times after initial infection, is independently at most 1 / 1000th lower than the amount of new virions or reporter signal observed from live recombinant AngHV-1 containing a functional ORF35, for example, at most 1 × 10⁻⁶ in ascending order of priority. 4 One in half, at most 1×10 5 One in half, at most 1×10 6 One in half, at most 1×10 7 One in half, at most 1×10 8 One in a fraction or at most 1×10 9 One in four. In the case of comparing wild-type and dysfunctional ORF35 strains that express the reporter protein, abortive infection can be particularly inferred when live recombinant AngHV-1 containing dysfunctional ORF35 does not produce any detectable amount of new virion or detectable amount of reporter gene expression.

[0065] In some implementations, the live recombinant AngHV-1 may contain at least one mutation in at least one gene other than ORF35.

[0066] In some embodiments, the live recombinant AngHV-1 may contain at least one mutation in at least one gene other than ORF35, wherein the gene contributes to virulence but is not essential for viral replication in cell cultures. The term "virulence" refers to the degree of pathogenicity of a virus, more specifically, the severity and harm of the disease caused by the virus after infection. High virulence is associated with a stronger ability to cause severe disease or death, while low virulence corresponds to a less severe manifestation of infection. Therefore, at least one mutation in such a gene can reduce viral virulence without significantly reducing the virus's ability to replicate in a host (particularly fish, and more particularly eels). The mutation may, for example, render such a gene at least partially or completely dysfunctional or defective, such as by causing a reduction or elimination of the expression or activity of the gene or its gene product. For example, the nature of the mutation may be as described elsewhere in this specification. In some embodiments, the at least one gene is selected from ORF5 encoding deoxyuridine triphosphatase, ORF25 encoding an interleukin-10 homolog, ORF77 encoding thymidine kinase, ORF101 encoding a TNFR family homolog, ORF124 encoding a TNFR family homolog, and combinations thereof.

[0067] In some embodiments, live recombinant AngHV-1 may contain at least one mutation in at least one gene other than ORF35, wherein the gene is essential for viral replication in cell cultures, and optionally may contain at least one mutation in at least one gene other than ORF35, wherein the gene contributes to virulence but is not essential for viral replication in cell cultures, thus providing a non-replicating form of the virus. "Non-replicating form" means that recombinant AngHV-1 retains the ability to infect cells or individual fish (e.g., particularly eels) but cannot replicate to the extent that it forms infectious progeny viruses. The mutation may, for example, render such a gene at least partially or completely dysfunctional or defective, such as by causing a reduction or elimination of the expression or activity of the gene or its gene product. For example, the nature of the mutation may be as described elsewhere in this specification. Such a non-replicating form of the virus can be cultured on a permissible cell line that stably expresses the functional form of the essential gene mutated in the virus (trans-complementation).

[0068] In some embodiments, the live recombinant AngHV-1, as taught herein, also comprises a heterologous nucleic acid. In some embodiments, the nucleic acid is DNA. In some preferred embodiments, the heterologous nucleic acid is contained within the genome of the recombinant AngHV-1. In this context, "heterologous" means a nucleic acid that is not native to or derived from AngHV-1, i.e., a non-AngHV-1 nucleic acid. In some embodiments, the heterologous nucleic acid may not be native to or derived from herpesviruses, i.e., a non-herpesvirus nucleic acid. The precise nature or sequence of the heterologous nucleic acid is not important, as long as it does not interfere with the characteristics of the live recombinant AngHV-1 relevant to the context of this invention, such as its ability to infect fish, particularly eels, or more particularly its ability to act as an AngHV-1 vaccine in fish, particularly eels. The size or length of the heterologous nucleic acid may vary, but should be chosen such that maintaining the full genome size facilitates packaging the genome within the viral particle.

[0069] Heteronucleotides can confer one or more useful or advantageous properties, characteristics, or activities on recombinant AngHV-1. Some non-limiting examples include biomarker properties, additional immunomodulatory properties (e.g., immunomodulatory properties against one or more non-AngHV-1 antigens), and / or adjuvant properties. In this respect, "biomarker properties" means that the heteronucleotide allows for direct or indirect differentiation between wild-type virus infection and vaccine virus infection (DIVA concept). Direct methods for differentiating between wild-type virus infection and vaccine virus infection may, for example, include PCR amplification using primer pairs designed to amplify sequences present only in the recombinant virus (e.g., using primers that hybridize in the wild-type genome and primers that hybridize in the transgene), but not in the natural AngHV-1 genome. Another direct method for differentiating between wild-type virus infection and vaccine virus infection may, for example, include visual detection of fluorescent protein biomarkers encoded by heteronucleotides but not expressed by wild-type AngHV-1. Indirect methods for distinguishing between wild-type viral infections and vaccine-prevented viral infections may include, for example, the use of an immune response with antibodies that specifically react with proteins encoded by heterologous nucleic acids but not with any proteins of the AngHV-1 wild-type virus.

[0070] In some embodiments, the heteronucleotide encodes a protein, particularly a non-AngHV-1 protein. In some embodiments, the heteronucleotide encodes an antigen of an eel pathogen other than AngHV-1 (e.g., a virus or microorganism), i.e., an immunogenic protein or protein fragment. In some non-limiting examples, the antigen may be the glycoprotein G of the eel virus X (EVEX) or an antigen of the aquatic double RNA eel virus European (EVE), or an immunogenic fragment thereof. When used in a vaccine, such a construct protects eels not only from AngHV-1 infection but also from EVEX and / or EVE infection (when both antigens are present). Suitable promoters for expressing heterologous genes in eukaryotic cells are widely known in the art. Some non-limiting examples of suitable promoters include the human cytomegalovirus (HCMV) early enhancer / promoter (HCMV IE), the EF-1α promoter, and the β-actin promoter.

[0071] On the other hand, nucleic acid molecules are provided that contain the genome of live recombinant AngHV-1 as taught herein. In some embodiments, the nucleic acid molecule is DNA. Methods for the extraction, purification, quantification, concentration, quality assessment, and storage of viral nucleic acids are well known in the art. For example, viral particles can be lysed using a lysis buffer typically containing detergent and optionally a protease; nucleic acids can be extracted by organic extraction with phenol-chloroform to separate nucleic acids from proteins, or by solid-phase extraction using silica membranes or magnetic beads, allowing for the washing away of contaminants and elution of nucleic acids in a low-salt buffer, or by binding of nucleic acids to silica membranes or magnetic beads under high-salt conditions; nucleic acids can optionally be further purified by additional washing steps or using a spinning column or magnetic beads specifically designed for purification; nucleic acids can be concentrated using alcohol precipitation or vacuum centrifugation; nucleic acids can be quantified using spectrophotometry (measuring absorbance at 260 nm) or fluorescence methods (using intercalating dyes); the quality and integrity of nucleic acids can be assessed using gel electrophoresis or other analytical methods; the isolated nucleic acids can typically be stored at -80°C or other suitable conditions, depending on subsequent use. When nucleic acid molecules containing the genome of live recombinant AngHV-1 are proliferated and / or genetically modified in host cells (e.g., in bacteria, yeast, or animal cells), similar steps can be applied with the necessary modifications to obtain the isolated form of nucleic acid.

[0072] Some other embodiments provide cells containing live recombinant AngHV-1 or its nucleic acid as taught herein. In some embodiments, the cells are bacterial cells, yeast cells, or animal cells. In some embodiments, the animal cells are fish cells, such as eel cells. In some embodiments, the cells are cells of a cell line. Any cells permitted to be infected with AngHV-1 are suitable for such purposes, including, in particular, eel cell lines mentioned elsewhere in this specification. Furthermore, exemplary host cells for maintaining, modifying, and / or proliferating the recombinant nucleic acid include, but are not limited to, bacterial cells (e.g., Escherichia coli, including strains BL21, DH5α, and JM109), yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris), and animal cells (e.g., Chinese hamster ovary (CHO) cells, Human Embryonic Kidney 293 (HEK293) cells, and COS cells (e.g., COS-7)).

[0073] On the other hand, pharmaceutical compositions (including veterinary pharmaceutical products) are provided that comprise live recombinant AngHV-1, nucleic acids, and / or cells as taught herein, and also comprise pharmaceutically acceptable carriers and / or adjuvants. The term "composition" generally refers to something consisting of two or more components, and more specifically refers to mixtures or blends of two or more materials (e.g., elements, molecules, substances, biomolecules, or microbial material), as well as reaction products and degradation products formed from the compositional materials. For example, a composition may comprise any pharmaceutical agent as taught herein combined with one or more other substances. For example, a composition may be obtained by combining (e.g., mixing) a pharmaceutical agent as taught herein with said one or more other substances. In some embodiments, the compositions of the present invention may be formulated as pharmaceutical compositions. Pharmaceutical compositions typically comprise one or more pharmacologically active ingredients (chemically and / or biologically active materials having one or more pharmacological effects) and one or more pharmaceutically acceptable carriers. As commonly used herein, compositions may be liquids, semi-solids, or solids, and may comprise solutions or dispersions.

[0074] In some embodiments, the pharmaceutical composition is a vaccine. As used herein, the term "vaccine" refers to a composition comprising an immunologically effective amount of one or more antigens, wherein said antigens are capable of inducing a specific immune response in a subject to prevent, improve, or treat a disease, particularly an infectious disease, and wherein said composition is formulated for administration via one or more routes suitable for inducing such an immune response. Vaccines can produce prophylactic or therapeutic immunity. An "immunologically effective amount" is an amount of antigen sufficient to elicit a desired immune response in a subject, which may include the production of antibodies, activation of lymphocytes, or the induction of other immune responses that contribute to the prevention, improvement, or treatment of a disease without causing significant adverse side effects. The term "therapeuticly effective amount" refers to an amount capable of eliciting a biological or medical response in a tissue, system, or animal sought by a researcher, veterinarian, physician, or other clinician, and particularly capable of preventing or alleviating one or more local or systemic symptoms or features of the disease or condition being treated. An appropriate therapeutically effective amount can be determined by a qualified physician or veterinarian, taking into full account the nature and severity of the disease condition and the age, size, and condition of the subject. The term “medicinal” as used herein, especially in relation to excipients and adjuvants, is consistent with the art and means compatible with other components of a pharmaceutical composition and harmless to its recipient.

[0075] The terms “carrier” or “excipient” as used herein are used interchangeably and broadly include any and all solvents, diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate, or phosphate buffer), solubilizers (e.g., Tween® 80, polysorbate 80), stabilizers, colloids, dispersion media, carriers, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aromatizers, thickeners, coating agents, antifungal agents, preservatives (e.g., Thimerosal™, benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), adjuvants, and building agents (e.g., lactose, mannitol). The use of such media and agents in the formulation of pharmaceutical compositions is well known in the art. Such substances should be non-toxic and should not interfere with the activity of the active ingredient. Acceptable carriers may include biocompatible, inert, or bioabsorbable salts, buffers, oligosaccharides or polysaccharides, polymers, viscosity modifiers, preservatives, etc. The exact properties of the carrier or other substance will depend on the route of administration, such as injection, immersion, or oral administration. In some embodiments, a pharmaceutically acceptable carrier can be as simple as water or a buffer solution.

[0076] As used herein, the term "adjuvant" refers to any substance that enhances a humoral or cellular immune response to an antigen. Adjuvants are typically used to achieve two objectives: slowing the release of the antigen from the site of application and / or stimulating the immune system. Common adjuvants known in the art are diverse in nature. Some non-limiting examples of adjuvants include mineral oils, vegetable oils, aluminum salts, saponins, beta-glucan, Freund's Complete Adjuvant, or polylactide-co-glycolide (PLGA) and other biodegradable polymers, cytokines, or other immunostimulants, and combinations thereof. Other non-limiting examples of adjuvants include, but are not limited to, hydrophilic adjuvants (e.g., aluminum hydroxide or aluminum phosphate) or hydrophobic adjuvants (e.g., mineral oil-based adjuvants, muramyl dipeptides, avidine, aluminum hydroxide, aluminum phosphate, oils, oil emulsions, saponins, dextran sulfate, glucan, cytokines, block copolymers, and immunostimulatory oligonucleotides). Some examples of adjuvants commonly used in fish farming include muramyl dipeptides, lipopolysaccharides, several dextrans and polysaccharides, and carbomer® (homogene). Suitable adjuvants are, for example, water-in-oil (w / o) emulsions, o / w emulsions, and w / o / w dual emulsions. Oil adjuvants suitable for w / o emulsions are, for example, mineral oils or metabolizable oils. Mineral oils include, for example, Bayol®, Marcol®, and Drakeol®; metabolizable oils include, for example, vegetable oils (e.g., peanut oil and soybean oil) or animal oils (e.g., fish oil squalane and squalene). Alternatively, vitamin E (tocopherol) solubilizers as described in EP382,271 may be advantageously used. A very suitable o / w emulsion is obtained, for example, from a 5% w / w to 50% w / w aqueous phase and a 95% w / w to 50% w / w oil adjuvant, more preferably using a 20% w / w to 50% w / w aqueous phase and an 80% w / w to 50% w / w oil adjuvant. The amount of adjuvant added depends on the properties of the adjuvant itself and the information provided by the manufacturer regarding such amounts.

[0077] When the recombinant AngHV-1, nucleic acid, or cells according to the invention are used as vaccine components for large-scale administration (e.g., by immersion, balneation, or oral administration), adjuvant administration is generally not required. However, if the vaccine formulation is injected directly into fish, the use of adjuvants is optional and may be preferred. Generally, formulations may include a variety of adjuvants to enhance the immune response, particularly in the case of formulations intended for injection.

[0078] Typically, vaccines are prepared as liquid solvents, emulsions, or suspensions for injection or delivery by immersion of fish in water. For example, liquid emulsions or emulsifiable concentrates can be prepared for addition to aquariums or bathtubs used for fishkeeping. Solid dosage forms (e.g., powders) suitable for dissolving or suspending in a liquid carrier prior to administration or for mixing with solid food can also be prepared. Vaccines can be ready-to-use lyophilized cultures for reconstitution with sterile diluents. For example, lyophilized cultures or cells can be reconstituted in 0.9% saline (optionally provided as part of the packaged vaccine product). A preferred formulation for injectable vaccines is an emulsion. Liquid or reconstituted vaccine forms can be diluted in small volumes (e.g., 1 to 100 volumes or other dilution ratios) of water before addition to a pen, container, or bathtub. In a preferred embodiment, vaccine formulations containing recombinant AngHV-1, nucleic acids, or cells as taught herein are in a dry form, such as powder, lyophilized form, compressed pellets, or tablets.

[0079] In some embodiments, recombinant AngHV-1 may be in the form of a tissue culture fluid. This fluid can be stored in the environment, preferably at -70°C or lower, and most preferably as a solution containing glycerol or another cryopreservative. In one specific example, the tissue culture fluid contains 20% v / v glycerol.

[0080] As taught in this article, recombinant AngHV-1, nucleic acids, or cells can be converted to a dry form by various methods. A particularly preferred drying method is lyophilization. Prior to the drying process, such as lyophilization, various components can be added to the culture medium, such as preservatives, antioxidants or reducing agents, and various excipients. Such excipients can also be added to the dried, for example, lyophilized virus after the drying step.

[0081] In a preferred embodiment, the vaccine according to the invention further comprises a stabilizer. The stabilizer can be added to the vaccine according to the invention, for example, to protect it from degradation, extend its shelf life, or improve freeze-drying efficiency. Available stabilizers are, in particular, SPGA (Bovarnik et al., 1950, J. Bacteriology, Vol. 59, p. 509), skim milk, gelatin, bovine serum albumin, carbohydrates (e.g., sorbitol, mannitol, trehalose, starch, sucrose, dextran, or glucose, lactose), proteins (e.g., albumin or casein or their degradation products), and buffers (e.g., alkali metal phosphates). Antibiotics such as neomycin and streptomycin can be added to prevent potential bacterial growth. Additionally, the vaccine may contain one or more suitable surfactants or emulsifiers, such as Span® or Tween®. The vaccine may also contain a so-called “carrier.” A carrier is a compound to which the AngHV-1 virus (in the form of viral particles or DNA) according to the invention attaches without covalently binding. Such carriers, particularly microcapsules, microalginates, liposomes, and macrosol, are all known in the art. A particular form of such carrier is Iscom. It goes without saying that the incorporation of other stabilizers, carriers, diluents, emulsions, etc., into the vaccine according to the invention is also within the scope of this invention. Such additives are described, for example, in well-known manuals, such as: “Remington: the science and practice of pharmacy” (2000, Lippincot, USA, ISBN: 683306472), and “Veterinary vaccinology” (edited by P. Pastoret et al., 1997, Elsevier, Amsterdam, ISBN: 0444819681).

[0082] When used in a vaccine in its dry form, recombinant AngHV-1, nucleic acid, or cells, as taught herein, may also contain a reconstitution fluid, preferably sterile water, saline, or physiological solution. It may also contain trace amounts of residual substances from the manufacturing process, such as cellular proteins, DNA, RNA, etc. While these substances are not additives themselves, they may still be present in the vaccine formulation.

[0083] Vaccines can be administered to fish alone, for example, through their feed or by forced oral administration, or by injection (e.g., via intramuscular or intraperitoneal routes). Alternatively, vaccines can be administered simultaneously to the entire fish population contained in a body of water by spraying, dissolving, and / or soaking. These methods can be used for vaccination of all species of fish (e.g., food fish and ornamental fish) and in a variety of environments, such as ponds, aquariums, natural habitats, and freshwater reservoirs.

[0084] Nucleic acid vaccines, especially DNA vaccines, containing the recombinant AngHV-1 genome as taught herein, can be readily administered via intradermal application, for example using a needle-free injector such as GeneGun®. This method of administration delivers the DNA directly into the cells of the animal to be vaccinated. In the pharmaceutical composition according to the invention, the preferred amount of the recombinant AngHV-1 DNA according to the invention is 10 pg to 1000 μg. Preferably, an amount of 0.1 to 100 μg is used. Alternatively, the fish can be immersed in a solution containing, for example, 10 pg to 1000 μg / ml of the DNA to be administered. All these techniques and methods of administration are well known in the art. Preferably, the vaccine according to the invention is formulated in a form suitable for injection or immersion vaccination (e.g., suspension, solution, dispersion, emulsion, etc.).

[0085] Dosing regimens for applying the vaccine according to the invention to target organisms can be single-dose or multi-dose administration, which can be given simultaneously or sequentially in a dose- and formulation-compatible manner and in amounts such as those required for immunogenicity. Those skilled in the art are fully capable of determining whether treatment is immunogenic, for example by experimentally challenging the vaccinated animal with infection and subsequently determining the clinical signs of disease, serological parameters, or by measuring the re-isolation of the pathogen in the target animal.

[0086] The preferred amount of a live vaccine containing the recombinant AngHV-1 viral strain according to the invention is expressed, for example, as plaque-forming units (pfu). For example, for a live viral vector, 1 to 1 × 10⁻⁶ units can be advantageously used. 10 Dosage range of plaque-forming units (pfu) per animal dose; preferably 1 × 10 2 Up to 1×10 6 The range of PFU / dosage is applicable. Many administration methods are possible, all known in the art. The vaccine according to the invention is preferably administered to fish by injection (intramuscular or intraperitoneal route), immersion, soaking, or oral administration. The administration protocol can be optimized according to standard vaccination practices. Preferably, the vaccine is administered by immersion. This is particularly effective when using such a vaccine in a commercial aquaculture environment.

[0087] Therefore, another aspect relates to live recombinant AngHV-1, nucleic acid, cell, or pharmaceutical composition as taught herein, for the immunization of fish against a herpesvirus. A method for immunizing fish against a herpesvirus is also provided, comprising administering an immunogenically effective amount of live recombinant AngHV-1, nucleic acid, cell, or pharmaceutical composition as taught herein. Optionally, the fish is an eel. Optionally, the herpesvirus is AngHV-1. Optionally, the fish is an eel, and the herpesvirus is AngHV-1.

[0088] In some embodiments, when live recombinant AngHV-1 contains a heterologous nucleic acid encoding an eel pathogen antigen other than AngHV-1, immunization is directed against said pathogen, or against AngHV-1 and said pathogen. In some embodiments, when live recombinant AngHV-1 contains a heterologous nucleic acid encoding glycoprotein G of the eel virus X (EVEX), immunization is directed against EVEX, or against AngHV-1 and EVEX. In some embodiments, when live recombinant AngHV-1 contains a heterologous nucleic acid encoding an EVE antigen, immunization is directed against EVE, or against AngHV-1 and EVE.

[0089] On the other hand, it relates to live recombinant AngHV-1, nucleic acid, cell, or pharmaceutical compositions as taught herein, for treating diseases caused by herpesviruses in fish. A method for treating diseases caused by herpesviruses in fish in need of treatment is also provided, comprising administering to the fish a therapeutically effective amount of a live recombinant AngHV-1, nucleic acid, cell, or pharmaceutical composition as taught herein. The term “treatment-required” or similar terms as used herein refers to a subject diagnosed with or suffering from the disease described herein and / or a subject seeking prevention of the disease. Optionally, the fish is an eel. Optionally, the herpesvirus is AngHV-1. Optionally, the fish is an eel, and the herpesvirus is AngHV-1. In some embodiments, the treatment is preventative. In some embodiments, the treatment is therapeutic. In some embodiments, the treatment is both preventative and therapeutic. In some embodiments, the fish is an eel, the herpesvirus is AngHV-1, and the treatment is preventative. In some embodiments, the fish is an eel, the herpesvirus is AngHV-1, and the treatment is therapeutic. Prophylactic use is intended to prevent infection, or at least the clinical manifestations of disease. Therapeutic use is for fish that have already contracted a disease caused by a herpesvirus.

[0090] In some embodiments, when live recombinant AngHV-1 contains a heterologous nucleic acid encoding an eel pathogen antigen other than AngHV-1, the disease is caused by said pathogen, or by AngHV-1 and said pathogen. In some embodiments, when live recombinant AngHV-1 contains a heterologous nucleic acid encoding glycoprotein G of the eel virus X (EVEX), the disease is caused by EVEX, or by AngHV-1 and EVEX. In some embodiments, when live recombinant AngHV-1 contains a heterologous nucleic acid encoding an EVE antigen, the disease is caused by EVE, or by AngHV-1 and EVE.

[0091] Live recombinant AngHV-1, as taught herein, can be readily produced using established cell culture techniques. Therefore, one aspect provides a method for producing recombinant live AngHV-1 infectious particles, the method comprising the steps of:

[0092] (a) Introducing live recombinant AngHV-1 or nucleic acid molecules as taught herein into permitted eukaryotic cells; and

[0093] (b) The cells were cultured to produce live recombinant AngHV-1.

[0094] Optionally, cell cultures or preferably cell supernatants containing live recombinant AngHV-1 can be used directly to prepare vaccine compositions, or the virus particles can be further purified from the cell cultures or preferably from the cell supernatants, as taught elsewhere in this specification.

[0095] While the invention has been described in conjunction with some specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be apparent from the foregoing. Therefore, it is intended to include all such alternatives, modifications, and variations as described below within the spirit and scope of the appended claims.

[0096] The aspects and embodiments of the invention disclosed herein are also supported by the following non-limiting examples.

[0097] Example

[0098] 1. Materials and methods used in the embodiments

[0099] Cells and AngHV-1 strain

[0100] As previously described, eel kidney (EK-1) cells were cultured (Donohoe et al., Genomes of Anguillid Herpesvirus 1 Strains Reveal Evolutionary Disparities and Low Genetic Diversity in the Genus Cyprinivirus. Microorganisms 2021, Vol. 9, 998). The AngHV-1 isolate used in this study (hereinafter referred to as the UK strain) was kindly provided by Dr. Keith Way (Centre for Environment, Fisheries, and Aquaculture Science, UK).

[0101] Recombinant AngHV-1 strains generated through eukaryotic cell recombination

[0102] The AngHV-1 UKLuc strain was generated in EK-1 cells via homologous directed recombination (HDR) using the UK strain as the parent strain. Figures 1A to 1D The LucGFP box consists of an EF1 promoter that drives the transcription of a bicistronic mRNA encoding a firefly luciferase Luc2 (LUC2) and copepod GFP (copGFP) protein linked by a T2A peptide. Figure 1C The cassette was inserted into the intergenic region ORF32 to ORF33 (MW580855.1) at coordinates 49134 to 49135 of the AngHV-1 genome to generate the UK Luc recombinant strain. The pGEMT LucGFP vector containing the recombinant fragment consists of a Luc cassette with 500 bp flanking sequences corresponding to the upstream (ORF32 end and ORF32 3'UTR) and downstream (ORF33 3'UTR and ORF33 end) regions of the insertion site. One day after transfection with the pGEMT LucGFP vector, EK-1 cells were infected with AngHV-1 at a multiplicity of infection (MOI) of 0.1 plaque-forming units (pfu) / cell. After 24 hours of incubation, cells expressing green fluorescence were sorted by flow cytometry using a FACS Aria (Becton Dickinson) system. GFP-positive cells were seeded onto a monolayer of EK-1 cells. Viral plaques expressing copGFP were selected and amplified twice to purify the AngHV-1 UK Luc recombinant.

[0103] Similarly, UK strain and UK Luc strain were used as parental strains to generate UK ORF35 Del and UK Luc ORF35 Del strains respectively in EK-1 cells via HDR. Figures 1A to 1D The pGEMT mCherry vector containing the recombinant fragment consists of an mCherry ORF, with each ORF flanked by a 500 bp sequence corresponding to the end of AngHV-1 ORF34 in the left homologous region and the intergenic region of ORF35 to ORF36 in the right homologous region, plus the end of ORF36. One day after transfection with the pGEMT mCherry vector, EK-1 cells were infected with AngHV-1 UK or UK-Luc strains at a multiplicity of infection (MOI) of 0.01 pfu / cell. Four days later, the supernatant was collected and diluted for infection of EK-1 cells. The infected cells were covered with a medium containing carboxymethyl cellulose (Donohoe et al., ibid.). Viral plaques expressing mCherry were selected and amplified until the inventors obtained plaques expressing 100% mCherry.

[0104] Genetic characteristics of AngHV-1 recombinants

[0105] The molecular structures of all recombinant strains were confirmed by monitoring SacI restriction fragment length polymorphisms via agarose gel electrophoresis and full-length genome sequencing, as previously described (Donohoue et al., ibid.).

[0106] Transcription analysis

[0107] EK-1 cells were either simulated with infection or infected with an MOI of 0.1 pfu / cell. 24 hours post-infection (pi), RNA was isolated using the NucleoSpin RNA Mini Kit (Macherey-Nagel), and residual DNA was removed using the TURBO DNA-Free Kit (Invitrogen). 5 µg of RNA was reverse transcriptase (RT) with Superscript III reverse transcriptase and oligomeric (dT) primers (Invitrogen) to produce cDNA. ORF32, ORF33, ORF34, ORF35, ORF36, and ORF55 (AngHV-1 DNA polymerase) were amplified using Phusion high-fidelity DNA polymerase (New England Biolabs) with the primer pairs listed in Table 1. PCR was performed with RT omitted to exclude amplification of contaminant viral genomic DNA from the purified RNA. Figure 2A, right image (RNA).

[0108] Table 1. Oligonucleotide primers

[0109]

[0110] * Coordinates are based on the reference AngHV-1 genome (GenBank accession number: MW580855.1).

[0111] **AngHV-1 sequence is underlined.

[0112] H1 and H2 in pGEMT H1.PmeI.H2 and pGEMT LucGFP correspond to the 500 bp end and start sequences of AngHV-1 ORF32 and ORF33, respectively.

[0113] H1 and H2 in pGEMT mCherry correspond to the 500 bp sequence at the end of ORF34 and the intergenic region between ORF35 and ORF36, respectively, plus the end of ORF36.

[0114] Italics: Sequences corresponding to PmeI restriction sites.

[0115] a Based on Rijsewijk et al., Development of a polymerase chain reaction for the detection of Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene. J Virol Methods 2005, Vol. 124: 87-94.

[0116] Indirect immunofluorescence staining

[0117] Cells were fixed at 4°C in phosphate-buffered saline (PBS) containing 4% (w / v) paraformaldehyde (PAF) for 15 min, followed by fixation at 20°C for 15 min. After washing with PBS, the samples were permeabilized at 37°C in PBS containing 0.1% (v / v) NP-40 for 15 min. Immunofluorescence staining (incubation and washing) was performed in PBS containing 10% fetal bovine serum (FCS) (v / v) (PBS-FCS). Rabbit polyclonal antibody (pAb) (1:2000 dilution) generated from purified AngHV-1 virions was used as the primary antibody. The primary antibody was incubated at 37°C for 1 h. After washing with PBS-FCS, Alexa Fluor 488 goat anti-rabbit immunoglobulin G (H+L) (Invitrogen) was used as the secondary antibody in PBS-FCS (1:1000 dilution). The secondary antibody was incubated at 37°C for 30 minutes. After washing with PBS-FCS, the fixed cells were incubated with PBS containing DAPI (1:2000 dilution) (ThermoFisher) for 5 minutes at room temperature, and then washed with PBS before mounting.

[0118] Virus growth assay

[0119] Triple cultures of EK-1 cells were infected with AngHV-1 at an MOI of 0.01 pfu / cell. After a 2-hour incubation period, the cells were washed with PBS and covered with culture medium. Cell supernatant was collected at consecutive intervals. After centrifugation at 900 g for 30 min at 4 °C to precipitate cell debris, the supernatant was collected and stored at -80 °C. Virus titration was performed by a triple plaque assay in EK-1 cells as previously described [1].

[0120] Viral plaque area measurement

[0121] As previously described (Donohoe et al., ibid.), viral plaque area determination was performed. Individual plaques were revealed by immunofluorescence staining and imaged using a Nikon A1R confocal microscope, and the area was measured using ImageJ software (Abràmoff et al., Image Processing with ImageJ. Biophotonics international 2004, Vol. 11, 36-42).

[0122] Virus photoinactivation by psoralen / UV treatment

[0123] To inactivate the UK ORF35 Del recombinant strain without affecting viral structural proteins (and thus its ability to enter cells susceptible to AngHV-1 infection), the virion was co-incubated with psoralen (4'-aminomethyltrimethylsalicylin hydrochloride, Sigma-Aldrich) and exposed to long-wave UV light (365 nm, 6 W, 0.16 A, UVPUVL-56, Analytica Jena US). In short, 1 × 10⁻⁶ virions were inactivated. 5 PFU UK ORF35 Del was incubated in 1 mL of L-15 medium containing 5 µg / mL psoralen and then incubated on ice at a distance of 5 cm from UV light in a 35 mm sterile plastic culture dish for 10 min. Total virus inactivation by psoralen / UV treatment was controlled by titrating residual infectious viral particles in vitro prior to in vivo experiments.

[0124] fish

[0125] European eels (Eriocheir sinensis) in their glass eel development stage were sourced from a certified commercial company (FoucherMaury, France). Microbiological, parasitic, and clinical examinations were conducted immediately upon arrival at the animal facility, followed by monthly monitoring to control fish health. The glass eels were grown in 40 L freshwater tanks maintained at 25°C. The fish were used at the yellow eel stage.

[0126] AngHV-1 vaccination pattern

[0127] Different inoculation methods were used in the study. mold Formula. (i) Inoculation by intraperitoneal (IP) injection. The eel was anesthetized by immersion in water containing benzocaine (25 mg / L water). 20 µl of insulin containing 2 × 10⁻⁶ mg / L was injected intraperitoneally using a 0.3 mL insulin syringe (BD Micro-Fine). 5 (ii) Inoculation by immersion in infectious water. Under continuous aeration, the fish were inoculated by immersion in water containing AngHV-1 (the dose used (pfu / mL) is described in the illustration) for 2 hours. (iii) Inoculation by intradermal infection. The eels were anesthetized by immersion in water containing benzocaine (25 mg / L water). Intradermal injection was performed using an electronic tattooing device. As part of the procedure, 20 µL of water containing 2 × 10⁻⁶ AngHV-1 was injected. 5The culture medium for PFU AngHV-1 was added to the sterile tattoo needle ink reservoir. Using the device, the inoculum was delivered intradermally at three points (all on the left side of the eel's body, including the anterior, middle, and posterior sections) along a linear pattern for approximately 1 cm. After the intradermal injection, the fish was placed in a recovery bath and then returned to its container.

[0128] Ethical Statement

[0129] The experimentation, maintenance, and care of the fish comply with the guidelines of the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes (CETS n◦123). Animal research has been approved by the local ethics committee of the University of Liège, Belgium (Laboratory Accreditation No. 1610008, Agreement No. 1896). All efforts have been made to minimize the suffering of the fish and improve their welfare.

[0130] Bioluminescence imaging

[0131] As previously described, IVIS (PerkinElmer) was used to image the luciferase activity of fireflies (Photinus pyralis) (Costes et al., The Major Portal of Entry of Koi Herpesvirus in Cyprinus Carpio Is the Skin. J. Virol. 2009, Vol. 83, 2819-2830; Boutier et al., Rational Development of an Attenuated Recombinant Cyprinid Herpesvirus 3 Vaccine Using Prokaryotic Mutagenesis and In Vivo Bioluminescent Imaging. PLOS Pathog 2015, Vol. 11, e1004690). For cell culture analysis, the medium was replaced with fresh medium containing D-luciferin (150 μg / mL) (Caliper LifeSciences). Analysis was performed after an incubation period of 10 minutes at room temperature. For in vivo analysis, fish were anesthetized with benzocaine (25 mg / L water). Fifteen minutes prior to bioluminescence analysis, D-luciferin (150 mg / kg body weight) was injected into the peritoneal cavity. In vivo analysis was performed with fish in their right and left lateral decubitus positions, and ex vivo analysis was performed after euthanasia with benzocaine (250 mg / L water). Dissected organs were analyzed independently of the body. All images were acquired using a maximum automatic exposure time of 1 minute, a pixel binning factor of 8, and an f / stop of 1. The relative intensity of transmitted light from bioluminescence was automatically determined using Living Image 4.7.3 software and represented as a pseudo-color image from purple (lowest intensity) to red (highest intensity). Regions of interest (ROIs) were manually plotted by tracing organ or body contours, and the mean radiance (p / s / cm²) was calculated. 2 The final measurement of bioluminescence emitted on the ROI was obtained as (photons / sec, p / sec). For skin, mean radiation was measured on both sides of the body, and the results for individual fish were expressed as the mean of both sides. For gills (mean of left and right gills) and visceral organs, ex vivo and separate analyses were performed. The positive cutoff value was the mean of values ​​obtained from fish with simulated infection (not shown in the figure) + 3 SD. For cell monolayers, the entire well was used as the ROI, and the photon flux was measured in photons / sec (p / sec).

[0132] Statistical analysis

[0133] First, the Shapiro-Wilk test (GraphPad Prism v8.0.1) was used to test the normality of the residuals for each dataset. The following comprehensive test was then performed on the data from virus growth, plaque size, and bioluminescence. Figure 2 C to 2E): Use two-way ANOVA (GraphPad Prism v8.0.1) for datasets with a Gaussian distribution or the Durbin test (Pohlert, T. PMCMR: Calculate Pairwise Multiple Comparisons of Mean Rank Sums 2018.; R Core Team R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, 2019). Post-hoc multiple comparisons between target groups were performed using the following methods: for Gaussian-distributed data, the Sidak test (for two groups) or paired Tukey test (for more than two groups) was used (Graphpad Prism v8.0.1), and for non-Gaussian-distributed data, the Wilcoxon test was used (R Core Team R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria 2019; Ogle, D., Doll, J., Wheeler, P., Dinno, A. FSA: Fisheries Stock Analysis. R package 2021; Ogle, D., Wheeler, P., Dinno, A. R package version 0.8.26.9000 2019). The variables used for each comprehensive test and their significance are described in the legend. IVIS measurements were compared using unpaired t-tests (two-tailed) or Mann-Whitney tests. Figure 3A , 3B Compare with 3B (continued), as appropriate. Use the Fisher-Pitman displacement test to compare the number of positive fish in each group among the strains. For Figure 4A , 4BThe IVIS measurements, as described in section 4B, were analyzed using the nonparametric Kruskal-Wallis test, followed by a two-stage step-up method (Graphpad Prism v8.0.1) to control for the false discovery rate (FDR). The Fisher-Pitman permutation test was used to compare the number of positive fish from each group with the simulated infection group. Results are shown in each corresponding graph, and statistical significance for all test results is indicated using the following symbols: *, p < 0.05; **p < 0.01; ***p < 0.001.

[0134] 2. Generation and characterization of AngHV-1 ORF35 deletion recombinant strains

[0135] Different AngHV-1 recombinants were generated in eukaryotic cells via homologous directed recombination (HDR). Figures 1A to 1D ).

[0136] Two parental AngHV-1 strains were used to generate AngHV-1 ORF35-deleted recombinants, namely the UK strain and the UK Luc recombinant strain derived from the former. Figures 1A to 1D Based on the efficient replication of the AngHV-1 UK strain in cell culture and its ability to induce AngHV-1 disease in yellow eels after inoculation with virus-containing water (unpublished data), it was selected as the parental strain. The recombinant UK Luc strain is derived from a UK strain obtained by inserting a luciferase / GFP bicistron reporter cassette (LucGFP cassette) into the intergenic region between the 3'UTR and ORF33 of ORF32. Figure 1C The molecular structure of the resulting recombinant UK Luc was controlled by full-length genome sequencing. RT-PCR showed that the insertion did not have a polar effect on the expression of ORF32 and ORF33. Figure 2 A). Examination of EK-1 cells infected with the UK Luc strain showed that both luciferase bioluminescence and GFP fluorescence were expressed. Figure 2B and 2B (Continued), thus demonstrating the function of the report box. Comparison of the parental UK strain and the derived recombinant UK Luc strain by viral growth assays and viral plaque area assays revealed that insertion of the LucGFP box had no effect on replication in cell culture. Figure 2(C, 2D, and 2E). Finally, a comparison of the virulence of UK and UK Luc strains by inoculating eels revealed no significant differences in morbidity and mortality (unpublished data). In summary, these results indicate that inserting the LuckFP box into the intergenic region between ORF32 and ORF33 has no significant effect on the biological characteristics of the UK strain.

[0137] Using UK and UK Luc strains as parental strains, ORF35-deleted recombinants were generated via HDR. Figure 1A and 1B The loss of ORF35 is induced by substitution of ORF35 via a sequence encoding the mCherry protein. Figure 1D This method allows for the identification of recombinant viral plaques. It generated UK ORF35 Del and UK Luc ORF35 Del recombinants. The molecular structure of these recombinants was controlled by PCR, restriction profiling analysis, and full-length genome sequencing (data not shown). The ability to generate AngHV-1 ORF35-deleted strains using this method indicates that ORF35 is not essential for viral replication in cell culture.

[0138] Next, to ensure that the mutation generated at the ORF35 locus did not affect the expression of flanking genes (potential polarity effect), the inventors compared the transcription of ORF34 and ORF36 in EK-1 cells after infection with the ORF35-deleted recombinant and its respective parental strains. Figure 2 A). RT-PCR analysis showed that the insertion had no significant effect on the expression of flanking genes. Figure 2 A, cDNA). The absence of contaminating viral genome in purified RNA samples was controlled by performing PCR reactions on samples where the reverse transcription step was omitted. Figure 2 A, RNA). No PCR product was detected. To confirm the expression of the expected reporter gene in the recombinant, the expression of luciferase bioluminescence, mCherry, and copGFP fluorescence in the two ORF35-deleted strains and their respective parental strains was analyzed. Figure 2B The results confirmed the expected pattern of reporter gene expression. Both the UK Luc and UK Luc ORF35 Del strains expressed luciferase bioluminescence. Figure 2B (left box) and copGFP fluorescence ( Figure 2B (Right box) Both, while mCherry fluorescence is associated with ORF35 deficiency ( Figure 2B (Right frame).

[0139] 3. ORF35 deficiency does not impair the production of AngHV-1 virions in cell culture.

[0140] Early studies have shown that the deletion of the ortholog of AngHV-1 ORF35 (ORF57) in CyHV-3 induces a decrease in virion production in cell culture (Boutier et al., 2015, ibid.; Boutier et al., Identification of an Essential Virulence Gene of Cyprinid Herpesvirus 3. Antiviral Res 2017, Vol. 145, 60-69). Here, the inventors analyzed whether the deletion of AngHV-1 ORF35 also leads to a decrease in virion production in cell culture. The replication kinetics and plaque size of the ORF35-deficient recombinant strains (UK ORF35 Del and UK Luc ORF35 Del strains) were compared with those of the parental strains (UK and UK Luc strains), as described in Materials and Methods. Interestingly, throughout the experiment, all recombinant strains achieved high and similar titers, and no significant statistical differences were detected between the different strains. Figure 2 C). Comparison of UK Luc ORF35 Del and UK Luc strains with bioluminescence measured at post-infection time further supports the conclusion that ORF35 deficiency has no negative effect on AngHV-1 viral replication in cell culture. Figure 2 E). No statistically significant differences were detected between the two strains at any study time point after inoculation. In summary, these results demonstrate that the deletion of ORF35 does not impair the production of AngHV-1 virions in cell culture. This result contrasts sharply with previous observations in CyHV-3 when orthologs of AngHV-1 ORF35 were missing.

[0141] Finally, the inventors also investigated the effect of ORF35 deletion on AngHV-1 plaque size in cell culture. Plaque area measurements revealed that plaques produced by the two ORF35 Del strains were significantly smaller than those produced by the wild-type parental strain (two-factor ANOVA, p < 0.0001 at 6 and 8 dpi). Post-hoc tests revealed that at 6 dpi, the plaque size of the wild-type UK strain was larger than that of the UKORF35 Del strain (p = 0.0120) and the UK Luc ORF35 Del strain (p = 0.0013). At 8 dpi, post-hoc tests revealed that the plaque size of the UK strain was significantly larger than that of the two recombinant ORF35 Del strains: UK ORF35 Del (p < 0.0001) and UK Luc ORF35 Del (p < 0.0001). It is noteworthy that at any point post-infection, there were no differences in viral titer or plaque size between UK and UK Luc, or between UK ORF35 Del and UK Luc ORF35 Del. Figure 2 (C and 2D). In summary, the above results indicate that ORF35 is not essential for AngHV-1 replication in cell culture, and that ORF35 deficiency does not impair virion production despite a slight reduction in viral plaque size.

[0142] 4. The effect of AngHV-1 ORF35 on viral replication in vivo

[0143] The above results indicate that the UK and UK Luc strains, as well as the UK ORF35 Del and UK Luc ORF35 Del, exhibit similar phenotypes in cell culture. Therefore, the inventors used UK Luc and UK Luc ORF35 Del to investigate the effect of ORF35 deficiency on the AngHV-1 biological cycle in vivo using bioluminescence imaging.

[0144] Three different inoculation methods were used to inoculate the yellow eel: intraperitoneal injection, immersion in virus-containing water, and intradermal inoculation. Figure 3A For intraperitoneal inoculation, use 2×10 5 PFU was injected intraperitoneally into each yellow eel, and the samples were subsequently analyzed by IVIS at 2, 4, 6, 8, and 10 dpi. Figure 3B(Left column). Infection in all tested organs peaked at approximately 8 dpi. Analysis of mean radioactivity in bilateral body, gills, brain, heart, and enterohepatic tissues using unpaired t-tests or Mann-Whitney tests (for each organ separately) revealed significant differences between the two strains in all organs, with the UK LucORF35 Del strain expressing no bioluminescent signal or expressing a much lower level of bioluminescent signal. Post-hoc analysis (signal intensity) and substitution tests (number of positive subjects) revealed significant differences between the strains. Until the end of the experiment, both the signal intensity and the number of positive subjects from the UK Luc strain were higher than those from the UK Luc ORF35 Del strain. The signal evolution observed in the UK Luc ORF35 Del strain indicates that, despite the use of an artificial systemic infection modality, inoculation still resulted in irregular and abortive infection.

[0145] Immersion inoculation allowed the inventors to examine the effect of ORF35 deletion on AngHV-1 infection in a host simulating natural infection. Eels were inoculated by immersion in water containing 4000 pfu / mL of UK Luc or UK Luc ORF35 Del strain. Figure 3A , 3B (Continued from 3B, middle column). Eels were analyzed by IVIS at 2, 4, 6, 8, and 10 dpi. As with IP inoculation, statistical analysis (for each organ separately) revealed significant differences between strains in both signal intensity and the number of positive subjects across all organs. These significant differences between strains, as observed with IP inoculation, were a result of the low or no signal expression of the UK LucDel ORF35 strain. Notably, when inoculated by immersion, no other positive signals were observed in the UKLuc ORF35 Del group except for one fish that tested positive at 2 dpi (low signal, observed only in the gills). In contrast, for the UK Luc group, at least half of the subjects tested positive in all organs at 6 dpi, and 100% of all organs tested positive at 10 dpi.

[0146] Social interactions between eels represent the primary transmission route of AngHV-1 between hosts, particularly through bites (undisclosed data). Therefore, the inventors aimed to simulate this transmission pattern using intradermal inoculation with an electronic tattooing device. For inoculation using this device, 20 µl (2 × 10⁻⁶) of... 5PFU (proton pumped ferrous sulfate) inoculum was added to the ink reservoir. The intradermal inoculum was then delivered to the anterior, middle, and posterior portions of the left side of each subject's body using the device. Using this method, the inoculum was delivered intradermally at multiple points along a linear pattern for approximately 1 cm. Individual eels were then analyzed by IVIS at 1, 2, 4, and 6 dpi. Figure 3B (Right column). Statistical analysis of each organ revealed that in all organs, the time of infection and the strain did not significantly affect the signal and number of positive subjects. However, at 4 and 6 dpi, the signal expressed by UKLuc in the skin was still higher than that of the UK Luc ORF35 Del strain.

[0147] Fish infected with either the UK Luc strain or the UK Luc ORF35 Del strain showed a positive signal at 2 dpi, but only in the skin. At 2 dpi, a very low positive signal close to the cutoff was also detected in the intestinal-hepatic tissue, but only for UK Luc ORF35 Del. Notably, no positive signal for UK Luc ORF35 Del was detected at a later time point in this organ. While these results provide some evidence that eels intradermally inoculated with either UK Luc or UK Luc ORF35 Del gradually develop systemic infection, no positive signal for UK Luc ORF35 Del was observed in the brain at any time point. As expected, the skin showed the strongest signal, and for both strains, the signal was predominantly co-localized at the intradermal inoculation site. However, in eels infected with UK Luc ORF35 Del, these signals decreased after 2 dpi. Figure 3B (right column), which corresponds to the healing and eventual disappearance of the lesion at the injection site.

[0148] In summary, these in vivo experimental results demonstrate that AngHV-1 ORF35 is an important virulence factor. Even after artificial inoculation via IP or intradermal injection, deletion of this gene still resulted in aborted replication cycles. Notably, inoculation of eels by immersion in virus-containing water resulted in no detectable IVIS infection in all but one subject.

[0149] 5. Testing the potential of the UK ORF35 Del strain as a candidate vaccine for large-scale vaccination of eels by immersion in virus-containing water.

[0150] In the final part of this study, the inventors tested the potential of the UK ORF35 Del strain as a candidate vaccine for large-scale vaccination of eels via immersion in virus-containing water. The ability of this strain to induce a protective immune response against wild-type challenge was tested using IVIS as follows (…). Figure 4AThe original yellow eel was immersed in UKORF35 Del (1×10⁻⁶) at different concentrations. 5 Vaccination was performed in water containing 75,000, 50,000, 25,000, and 5,000 pfu / mL. Simultaneously, a group of eels was exposed to a higher test dose (corresponding to 1 × 10⁻⁶ pfu / mL before inactivation) that had been inactivated by psoralen / UV. 5 Viri bodies at pfu / mL). Psoralen / UV inactivation relies on DNA cross-linking, affecting DNA replication and transcription, but preserving the structure and proteins of the viral particles, thus retaining the virus's ability to bind and enter host cells (Elveborg et al., Methods of Inactivation of Highly Pathogenic Viruses for Molecular, Serology or Vaccine Development Purposes. Pathogens 2022, Vol. 11, 271). A group of eels were also simulated to be infected during primary infection ( Figure 4A , 4B (Continued from 4B, simulation). Eels were challenged with the AngHV-1 UK Luc strain (4000 pfu / mL) by immersion exposure 37 days after the initial inoculation, and analyzed by IVIS at 3, 6, 9, 12, and 15 dpi. Figure 4A , 4B (Continued from 4B).

[0151] Eels that were simulated and challenged during the initial inoculation exhibited normal infection dynamics. Figure 4B and 4B continued (Right column of each organ segment). On day 3 post-challenge, one-third of the challenged eels expressed bioluminescent signals in their skin and / or gills, and only one eel expressed bioluminescent signals in its visceral organs (brain and heart). At 6 dpi and later time points, all eels from the simulated infection group showed infection not only in their skin and gills but also in other tested organs (brain, heart, and gut-liver). These data suggest that the challenge used in this study resulted in systemic infection in all unvaccinated subjects.

[0152] Primary exposure to infectious particles of the UK ORF35 Del strain reduced replication of the UK Luc strain in a dose-response manner. Figure 4B and 4B continued By performing statistical analysis on each organ across all time points between groups to compare radiation signal (p(rad)) and the number of positive fish (p(no)) Figure 4B and 4B continuedThese analyses showed that all tested doses of the UK ORF35 Del strain infectious particles resulted in a significant reduction in the number of positive fish. This conclusion was drawn after considering all tested organs. Statistical analysis of signal radiation also supports the ability of the UK ORF35 Del strain to induce a protective immune response against wild-type challenge. Significant effects were observed at all doses tested on the skin and gills, while significant effects were observed on the brain, heart, and gut-liver at doses of 50,000 pfu / mL or higher.

[0153] In summary, the results of these experiments support the potential of the UK ORF35 Del strain as a candidate vaccine against AngHV-1 for large-scale vaccination of eels via immersion in water containing the infectious virus. Notably, vaccination of fish at doses of 100,000 and 50,000 pfu / mL resulted in no detectable positive fish (regardless of post-challenge time or the organ tested). At a dose of 75,000 pfu / mL, only three fish showed mild positivity (day 9, heart; day 12, gills and brain).

[0154] Figure 3A , 3B And 3B continuation (abortive infection following inoculation via immersion in infectious water) and Figure 4A , 4B The results shown in 4B (the ability of the virus to confer protective immunity against wild-type challenges) appear to suggest that vaccination with the UK ORF35 Del strain may be independent of viral infectivity and function like an inactivated vaccine. However, the efficacy of psoralen-UV inactivated particles with UK ORF35Del ( Figure 4B and 4B continued Results obtained from fish vaccinated with psoralen / UV (100,000 pfu / mL) ruled out this hypothesis. The fish in this group showed similar infection with the wild-type UK Luc strain as the simulated vaccination group. No statistically significant differences were observed between the two groups in terms of radiation signal or the number of positive fish in any of the organs tested.

[0155] In summary, the results of this study indicate that the absence of AngHV-1 ORF35 does not affect the production of virions in cell culture, but it does induce abortive infection in vivo after inoculation with virus-containing water. Unexpectedly, the inventors observed that this abortive infection is both necessary and sufficient to induce a protective immune response against wild-type challenge.

Claims

1. Live recombinant eel herpesvirus 1 (AngHV-1) containing a dysfunctional open reading frame 35 (ORF35) genome.

2. The live recombinant AngHV-1 according to claim 1, wherein ORF35 is completely or partially missing.

3. The live recombinant AngHV-1 according to claim 1 or 2, which induces abortive infection in eels (Anguilla).

4. The live recombinant AngHV-1 according to any one of claims 1 or 2, comprising at least one mutation in at least one gene other than ORF35, wherein said gene contributes to virulence but is not essential for viral replication in cell cultures.

5. The live recombinant AngHV-1 according to claim 4, wherein the gene is selected from ORF5 encoding deoxyuridine triphosphatase, ORF25 encoding an interleukin-10 homolog, ORF77 encoding thymidine kinase, ORF101 encoding a TNFR family homolog, ORF124 encoding a TNFR family homolog, and combinations thereof.

6. The live recombinant AngHV-1 according to any one of claims 1 or 2, further comprising heterologous nucleic acid.

7. The live recombinant AngHV-1 according to claim 6, wherein the heterologous nucleic acid encodes an antigen of an eel pathogen other than AngHV-1, such as glycoprotein G of the rhabdovirus eel virus X (EVEX) or an antigen of the aquatic double RNA virus European eel virus (EVE), or an immunogenic fragment thereof.

8. A nucleic acid molecule comprising the genome of the live recombinant AngHV-1 as defined in any one of claims 1 to 7, optionally wherein the nucleic acid molecule is DNA.

9. A cell comprising live recombinant AngHV-1 as defined in any one of claims 1 to 7 or comprising nucleic acid as defined in claim 8, optionally wherein the cell is a bacterial cell, a yeast cell or an animal cell, optionally wherein the animal cell is a fish cell, such as an eel cell.

10. A pharmaceutical composition comprising live recombinant AngHV-1 as defined in any one of claims 1 to 7, nucleic acid as defined in claim 8, and / or cells as defined in claim 9, and further comprising a pharmaceutically acceptable carrier and / or adjuvant, optionally wherein said pharmaceutical composition is a vaccine.

11. Use of the live recombinant AngHV-1 as defined in any one of claims 1 to 7, the nucleic acid as defined in claim 8, the cell as defined in claim 9, or the pharmaceutical composition as defined in claim 10 in the preparation of a medicament for immunization of fish, wherein the fish is optionally an eel, wherein: i) The immunization is against herpesvirus, optionally against AngHV-1; ii) When the live recombinant AngHV-1 contains a heterologous nucleic acid encoding an antigen of an eel pathogen other than AngHV-1, the immunization is against the pathogen, or the immunization is against both AngHV-1 and the pathogen; iii) When the live recombinant AngHV-1 contains a heterologous nucleic acid encoding glycoprotein G of the rhabdovirus eel virus X (EVEX), the immunization is against EVEX, or the immunization is against both AngHV-1 and EVEX; or iv) When the live recombinant AngHV-1 contains a heterologous nucleic acid encoding an EVE antigen, the immunization is against EVE, or the immunization is against both AngHV-1 and EVE.

12. Use of the live recombinant AngHV-1 as defined in any one of claims 1 to 7, the nucleic acid as defined in claim 8, the cell as defined in claim 9, or the pharmaceutical composition as defined in claim 10 in the preparation of a medicament for treating a disease in fish, optionally eels, wherein the treatment comprises preventive and / or therapeutic treatment, wherein: i) The disease is caused by a herpesvirus, optionally targeting AngHV-1; ii) When the live recombinant AngHV-1 contains a heterologous nucleic acid encoding an antigen of an eel pathogen other than AngHV-1, the disease is caused by the pathogen, or the disease is caused by AngHV-1 and the pathogen; iii) When the live recombinant AngHV-1 contains a heterologous nucleic acid encoding glycoprotein G of the rhabdovirus eel virus X (EVEX), the disease is caused by EVEX, or the disease is caused by AngHV-1 and EVEX; or iv) When the live recombinant AngHV-1 contains a heterologous nucleic acid encoding an EVE antigen, the disease is caused by EVE, or the disease is caused by AngHV-1 and EVE.

13. A method for generating recombinant live AngHV-1 infectious particles, the method comprising the following steps: (a) Introducing a live recombinant AngHV-1 as defined in any one of claims 1 to 7 or a nucleic acid molecule as defined in claim 8 into a permitted eukaryotic cell; and (b) The cells are cultured to produce the live recombinant AngHV-1.

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