WO2018029301A1 - Nouveau virus pathogène de poisson - Google Patents

Nouveau virus pathogène de poisson Download PDF

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WO2018029301A1
WO2018029301A1 PCT/EP2017/070336 EP2017070336W WO2018029301A1 WO 2018029301 A1 WO2018029301 A1 WO 2018029301A1 EP 2017070336 W EP2017070336 W EP 2017070336W WO 2018029301 A1 WO2018029301 A1 WO 2018029301A1
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virus
gene
seq
dna
herpesvirus
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PCT/EP2017/070336
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English (en)
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Siow Foong CHANG
Kah Sing NG
Luc Grisez
Ad DE GROOF
Wannes VOGELS
Lia VAN DER HOEK
Martin DEIJS
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Intervet International B.V.
Intervet Inc.
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Priority to CN201780049432.3A priority Critical patent/CN109890409A/zh
Priority to SG11201900912PA priority patent/SG11201900912PA/en
Publication of WO2018029301A1 publication Critical patent/WO2018029301A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention pertains to a novel fish pathogenic virus causing disease in fish, to cell cultures comprising said virus, DNA fragments and corresponding proteins of the said virus, to vaccines on the basis of said virus, DNA and/or protein and to antibodies reactive with said virus and diagnostic test kits for the detection of said virus.
  • Affected fish have generalized skin lesions, become lethargic, and show pronounced inappetence. As the disease progresses, the skin lesions become more severe, leading to a darkening of the skin with pale whitish patches and erosions of the fins and tail giving a ghost-like appearance to the fish. Eyes become swollen and slightly cloudy. The internal signs of the disease often noticed are enlarged spleen and kidneys. Kidneys become friable and easily detachable. Some pallor of the liver can often be observed. The gills become pale as the disease progresses.
  • the causative agent of the disease as described here above is a herpesvirus and a member of the family of Alloherpesviridae. It is an icosahedral virus belonging to the double-stranded DNA-viruses. The virus bears a certain albeit low level of resemblance to viruses of the family Alloherpesviridae, a family of herpesviruses that is pathogenic for fish or amphibians. Viruses of the Alloherpesviridae are enveloped, with icosahedral and spherical to pleomorphic geometries. Their diameter is around 150-200 nm. Genomes are linear and non-segmented, between about 100-250 kbp in length.
  • the virus of the present invention has a genome of about 130 kbp.
  • Figure 1 shows a phylogenetic tree indicating the relation between the known Alloherpesviridae and the newly found virus. The new virus is referred to in this tree as Lates Calcarifer Herpes Virus (LCHV).
  • LCHV Lates Calcarifer Herpes Virus
  • MEGA5 Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher, Masatoshi Nei and Sudhir Kumar. Mol. Biol. Evol. 28(10): 2731-2739. 201 1 doi:10.1093/molbev/msr121 Advance Access publication May 4, 201 1 ).
  • Alloherpesviridae more specifically the Alloherpesviridae found in fish are summarised in a review paper by Hanson, L. et al., (Viruses 3: 2160-2191 , 201 1 ).
  • the herpesvirus according to the invention was found in Asian sea bass (Lates calcarifer). So far no herpesviruses have been described that are pathogenic to Asian sea bass. It cannot be excluded that the virus is pathogenic for other (sub)-tropical fish as well.
  • the Ictalurid herpesvirus 1 IHV1
  • This virus is pathogenic for channel catfish.
  • the overall sequence identity between the virus according to the invention and the Ictalurid herpesvirus 1 at the nucleotide level is well below 60%.
  • the sequence identity with the more distantly related Anguilid Herpesvirus (AHV) and Koi Herpesvirus (KHV) is lower.
  • Table 1 shows a comparison between several of the genes identified in the new herpesvirus and the homologous genes in IHV1.
  • the table shows the most important ORF's identified (left hand column), with the proviso that identified ORF's having less than 300 base pairs are not taken up in the sequential numbering (but simply ignored for the purposes of this table), the position on the DNA (for one representative example of the virus, deposited at Institut Pasteur, see below), the corresponding ORFs in IHV1 , and the level of sequence identity at the amino acid level with these known ORFS has been indicated in the table.
  • ORF open reading frame (number) in LCHV
  • AA length length of the amino acid hit in BLAST search
  • IHV Id. percentage AMINOACID identity between LCHV ORF and corresponding IHV ORF IHV-1 : lctalurid Herpes Virus - 1
  • Homologue Putative function of the protein encoded by the LHCV ORF based on homology with known proteins
  • the wild type form of the current virus is the virus in a replication competent form, as can be isolated from diseased fish, in particular Asian sea bass, and capable of inducing the same disease in healthy fish of the species of fish from which the virus in its wild type form was isolated.
  • a wild type virus after being inactivated or attenuated, per definition, is capable of inducing disease in its wild type form.
  • An isolated virus is a virus set free from tissue with which the virus is commonly associated in a diseased host in nature and transferred free from other viruses and bacteria to a vessel such as a dish, flask or bio-reactor.
  • An example of an isolated virus is a virus present in a cell culture of a specific cell line in a bioreactor.
  • An isolated DNA fragment is a DNA fragment that is taken out of its natural (whole) DNA as present in the corresponding, natural occurring replication competent organism. Such DNA fragment may be present as such in a stabilising fluid, or may be recombinantly transferred into DNA of another organism. In each case, the DNA fragment is still isolated in the sense of the present invention.
  • An isolated protein is a protein that is taken out of its natural environment, i.e. out of its natural association with the corresponding natural occurring replication competent organism.
  • a vaccine is a pharmaceutical composition that is safe to administer to a subject animal, and is able to induce protective immunity in that animal against a pathogenic micro- organism, i.e. to induce a successful prophylactic treatment.
  • a successful prophylactic treatment in this sense is a treatment aiding in preventing or ameliorating an infection with that pathogen or a disorder arising from that infection, resulting from a post treatment challenge with the pathogenic pathogen, in particular to reduce its load in the host after such challenge and optionally to aid in preventing or ameliorating one or more clinical manifestations resulting from the post treatment infection with the pathogen.
  • ORF open reading frame
  • the novel virus is characterised as an isolated herpesvirus which is a member of the family of Alloherpesviridae, and which in its wild-type form causes a disease in Asian sea bass which is characterised by the following signs (after intraperitoneal challenge with the virus in a laboratory environment): onset of clinical signs around 3 days after challenge, generalized skin lesions (in a significant amount, typically over 50%, of the cases) leading to darkened skin with pale patches and fin erosions, lethargy with observed loss of swimming equilibrium ("observed” meaning that it is seen but not in all cases, only occuring with extremely lethargic fish), (almost complete) inappetence, increase in opercular respiration rate, and occurrence of mortalities about 2 weeks after challenge.
  • the virus has DNA that corresponds to DNA having a sequence according to SEQ ID NO:1 .
  • the level of identity over the full length of this DNA is over 70%, preferably over 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, over 99% or even up to 100%. It is noted that it is currently believed that around position 80,000 (out of the 130k base pairs) the virus has an internal repeat.
  • the virus can is present in four genome isomers (as explained by Mahiet et al. in: Structural variability of the Herpes Simplex Virus 1 genome In Vitro and In Vivo; Journal of Virology, august 2012, Volume 86, Number 16, pp 8592 - 8601 ). It may be that the novel virus has one or more additional internal repeats besides the repeat around bp 80,000. However, this has not been established yet. SEQ ID N0:1 corresponds to one of the possible genome isomers of the novel virus.
  • At least 95% of the open reading frames in its DNA, at least 95% of the open reading frames (in particular the ORF's comprising at least 300 basepairs), for example at least 96%, 97%, 98%, 99% or 100%, have at least 80% sequence identity, for example at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% identity, with the corresponding open reading frames of the DNA having a sequence according to SEQ ID NO:1 .
  • a suitable program for the determination of a level of identity is the nucleotide blast program (blastn) of NCBI's Basic Local Alignment Search Tool, using the "Align two or more sequences" option and standard settings (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
  • the isolated herpesvirus which is a member of the family of Alloherpesviridae, has at least one of the identifying characteristics of the virus deposited under Accesion Number CNCM 1-5118 at the Collection Nationale de
  • the virus can be identified as the novel alloherpesvirus according to the present invention, i.e. Lates Calcarifer Herpes Virus.
  • the identifying characteristics are chosen from the group consisting of 1 ) the virus having a DNA sequence that over its full length is at least 70% (or at least 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) identical to the sequence according to SEQ ID NO:1 , with the above mentioned proviso regarding the internal repeat; or 2) the virus in its wild type form causes disease in Asian sea bass which is characterised by the following signs: onset of clinical signs around 3 days after challenge, generalized skin lesions (
  • the virus is characterised in that the virus has DNA that corresponds to DNA having a sequence according to SEQ ID NO:1 .
  • the virus has DNA that over its full length is at least 70% identical to the DNA according to SEQ ID No.1 (with the above proviso regarding the internal repeat).
  • the level of identity may be higher, for example 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100%.
  • the virus is characterised in having DNA wherein at least 95% of the open reading frames (in particular the ORF's comprising at least 300 base pairs), for example at least 96%, 97%, 98%, 99% or 100%, have at least 80% sequence identity, for example at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% identity, with the corresponding open reading frames of the DNA having a sequence according to SEQ ID NO:1 .
  • the novel virus can be distinguished from known members of the Alloherpesviridae on the basis of the coding DNA sequences of its Major Envelope Protein (ORF28) and its dUTPase (ORF1 ). It turned out that the Major Envelope Protein of the virus has a level of sequence identity with the MEP of even the nearest of the other species of the Alloherpesviridae of only 30%.
  • the dUTPase has a level of sequence identity of only 45% with the nearest dUTPase of the other species of the Alloherpesviridae.
  • Typical examples of the DNA sequence encoding the MEP and dUTPase are shown in SEQ ID NO: 2 and 4 respectively.
  • a protein that is either a MEP or dUTPase according to the invention has a sequence identity of at least 70% to the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:5 respectively, thus having a sequence identity of 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% with these sequences.
  • the embodiments (A-G) relating to these proteins (i.e. MEP and dUTPase) in particular thus are as follows:
  • A an isolated herpesvirus comprising a Major Envelope Protein (MEP) gene, characterized in that the virus is a member of the family Alloherpesviridae, the virus causes disease in Asian sea bass, and the nucleotide sequence of the MEP gene has a level of identity of at least 80% (for example 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence as depicted in SEQ ID NO: 2.
  • MEP Major Envelope Protein
  • B an isolated herpesvirus comprising a dUTPase gene, characterized in that the virus is a member of the family Alloherpesviridae, the virus causes disease in Asian sea bass, and the nucleotide sequence of the dUTPase gene has a level of identity of at least 80% (for example 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence as depicted in SEQ ID NO: 4.
  • C an isolated herpesvirus having both the MEP gene and the dUTPase gene, characterized in that the nucleotide sequence of the MEP gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 2 and the nucleotide sequence of the dUTPase gene has a level of identity of at least 80% (for example 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence as depicted in SEQ ID NO: 4.
  • D an isolated herpesvirus comprising a Major Envelope Protein (MEP) gene, characterized in that the virus is a member of the family Alloherpesviridae, the virus causes disease in Asian sea bass, and the MEP gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 21 and 22 to give a PCR product of 277 +/- 10 base pairs.
  • MEP Major Envelope Protein
  • E an isolated herpesvirus comprising a dUTPase gene, characterized in that the virus is a member of the family Alloherpesviridae, the virus causes disease in Asian sea bass, and the dUTPase gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 23 and 24 to give a PCR product 346 +/- 10 base pairs.
  • F an isolated herpesvirus comprising the MEP gene and the dUTPase gene, characterized in that the MEP gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 21 and 22 to give a PCR product of 277 +/- 10 base pairs and the dUTPase gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 23 and 24 to give a PCR product of 346 +/- 10 base pairs.
  • G an isolated herpesvirus, characterized in that the nucleotide sequence of the MEP gene has a level of identity of at least 80% (or at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence as depicted in SEQ ID NO: 2 and the nucleotide sequence of the dUTPase gene has a level of identity of at least 80% (or at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence as depicted in SEQ ID NO: 4 and in that the viral DNA reacts in a PCR reaction with a primer set as depicted in SEQ ID NO
  • the embodiments D through G make use of a PCR-test using primer sets for the Major Envelope Protein gene sequence or the dUTPase gene sequence of a virus according to the invention.
  • Two different primer sets of which the sequence is depicted in SEQ ID NO: 21 -22 and SEQ ID NO: 23-24 were elected.
  • the PCR-test using the first primer set (SEQ ID NO: 21 -22) that reacts with the Major Envelope Protein gene of the virus uses the two primers LCHV MEP FW and LCHV MEP REV (see table 2b in the examples section).
  • the PCR-test using the second primer set reacts with the dUTPase gene of the virus and uses the two primers LCHV dUTP FW and LCHV dUTP REV (see Table 2c in the examples section).
  • the tests which are described in more detail in the Examples section, are standard PCR tests. If analysis of the PCR-product of the first primer set reveals a PCR product of approximately 277 base pairs or if analysis of the PCR-product of the second primer set reveals a PCR product of approximately 346 base pairs, and the virus is a member of the Alloherpesviridae and causes disease in Asian sea bass, this unequivocally demonstrates that the analysed virus is a virus according to the invention.
  • a PCR product of approximately 277 base pairs is a PCR product with a length of between 277 + 10 and 277 - 10 base pairs.
  • a PCR product of approximately 346 base pairs is a PCR product with a length of between 346 + 10 and 346 - 10 base pairs.
  • H-K relating to other proteins specific for the novel virus, i.e. a Terminase and a Polymerase, in particular are as follows:
  • H an isolated herpesvirus comprising a Terminase gene, characterized in that the virus is a member of the family Alloherpesviridae, the virus causes disease in Asian sea bass, and the Terminase gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 25 and 26 to give a PCR product of 585 +/- 10 base pairs.
  • I an isolated herpesvirus comprising a Polymerase gene, characterized in that the virus is a member of the family Alloherpesviridae, the virus causes disease in Asian sea bass, and the Polymerase gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 27 and 28 to give a PCR product 314+/- 10 base pairs.
  • Terminase gene an isolated herpesvirus comprising the Terminase gene and the Polymerase gene, characterized in that the Terminase gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 25 and 26 to give a PCR product of 585+/- 10 base pairs and the Polymerase gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 27 and 28 to give a PCR product of 314 +/- 10 base pairs.
  • K an isolated herpesvirus, characterized in that the nucleotide sequence of the
  • Terminase gene has a level of identity of at least 80% (or at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence as depicted in SEQ ID NO: 6 and the nucleotide sequence of the polymerase gene has a level of identity of at least 80% (or at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence as depicted in SEQ ID NO: 8 and in that the viral DNA reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 25 and 26 to give a PCR product of 585 +/- 10 base pairs and reacts
  • the embodiments H through K make use of a PCR-test using primer sets for the Terminase gene sequence or the Polymerase gene sequence of a virus according to the invention.
  • Two different primer sets of which the sequence is depicted in SEQ ID NO: 25-26 and SEQ ID NO: 27-28 were elected.
  • the PCR-test using the first primer set (SEQ ID NO: 25-26) that reacts with the Terminase gene of the virus uses the two primers LCHV TER FW and LCHV TER REV (see table 2d in the examples section).
  • the PCR-test using the second primer set (SEQ ID NO: 27-28) reacts with the
  • Polymerase gene of the virus uses the two primers LCHV POL FW and LCHV POL REV (see Table 2e in the examples section).
  • the tests which are described in more detail in the Examples section, are standard PCR tests. If analysis of the PCR-product of the first primer set reveals a PCR product of approximately 585 base pairs or if analysis of the PCR-product of the second primer set reveals a PCR product of approximately 314 base pairs, and the virus is a member of the Alloherpesviridae and causes disease in Asian sea bass, this unequivocally demonstrates that the analysed virus is a virus according to the invention.
  • L a (isolated) DNA fragment comprising a gene encoding a Major Envelope Protein characterized in that said gene has a level of identity of at least 80% (or at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence of the MEP gene as depicted in SEQ ID NO: 2, and M: a (isolated) Major Envelope Protein encoded by this DNA fragment.
  • N a (isolated) DNA fragment comprising a gene encoding an dUTPase characterized in that said gene has a level of identity of at least 80% (or at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) to the nucleotide sequence of the dUTPase gene as depicted in SEQ ID NO: 4, and O: a (isolated) dUTPase encoded by this DNA fragment.
  • genes could also be useful in identifying the novel virus according to the invention.
  • One of these genes is the gene corresponding to ORF224 (SEQ ID NO:10), which encodes for a membrane (glyco)protein (SEQ ID NO:1 1 ).
  • Yet another gene is the gene homologous to the lctalurid herpes virus 1 (IHV1 ) TK (Thymidine kinase) gene. In IHV-1 this gene corresponds to ORF5 (Hanson et al., Virology. 1994 Aug 1 ; 202(2):659-64).
  • IHV1 lctalurid herpes virus 1
  • TK Thimidine kinase
  • Deoxyribonucleoside kinase with an identity of 33% at the amino acid level with ORF 5 of IHV-1 .
  • Another gene (SEQ ID NO:12) for identifying the novel virus is the gene comprising ORF206, coding for a Major Capsid Protein (SEQ ID NO:13).
  • Still another embodiment according to the present invention pertains to any (isolated) DNA fragment having an open reading frame having a length of at least 100
  • nucleotides wherein the DNA has at least 80% (or at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100%) sequence identity with an open reading frame of the DNA that has a sequence according to SEQ ID NO:1 .
  • a length of 30-40 nucleotides has found to be sufficient to distinguish the DNA of a virus according to the invention over the DNA of any virus publicly known
  • a practical relevant length, in particular for corresponding sub-unit vaccines based on the corresponding protein is at least 100 nucleotides (or at least 150, 200, 250 or even at least 300 nucleotides) so as to correspond to a protein having a relevant and distinguishing 3D conformity that corresponds to relevant immunogenic epitopes of a viral protein.
  • the invention also pertains to a (isolated) protein encoded by such a DNA fragment.
  • the invention also pertains to a cell culture (i.e an artificial culture of a restricted number of types of cells, also called a cell line, in an artificial culture vessel; also described a process by which cells are grown under controlled conditions outside of their natural environment) comprising the novel virus according to the invention in a replication competent form.
  • a cell culture i.e an artificial culture of a restricted number of types of cells, also called a cell line, in an artificial culture vessel; also described a process by which cells are grown under controlled conditions outside of their natural environment
  • a cell line i.e an artificial culture of a restricted number of types of cells, also called a cell line, in an artificial culture vessel; also described a process by which cells are grown under controlled conditions outside of their natural environment
  • a cell line i.e an artificial culture of a restricted number of types of cells, also called a cell line, in an artificial culture vessel; also described a process by which cells are grown under controlled conditions outside of their natural environment
  • Several fish cell lines are potentially useful to support the replication of a
  • the invention pertains to a vaccine for combating herpesvirus disease in fish, wherein said vaccine comprises a herpesvirus according to the invention, or an immunogenic protein as described here above and a
  • a carrier may be as simple as water, as long as it is suitable for administration of the material in clinically relevant amounts without causing unacceptable side effects.
  • Typical carriers are emulsions of oil and water, suspensions of insoluble adjuvants (typically aluminium or other salts, or large immune stimulating polymeric molecules) in water or solutions of soluble adjuvants such as saponins, PAMP's, carbopol or other immune stimulating molecules.
  • the carrier comprises stabilisers and preservatives as is commonly known in the art.
  • the vaccine comprises a herpesvirus according to the invention in a live attenuated (i.e.
  • Vaccines consisted of peptides, (recombinant) viral proteins, mixtures of viral proteins, whole and fractionated killed virus (see for example Yasumoto, S. et al. in Fish Pathology 41 : 141 -145 (2006) which describes a vaccine comprising a inactivated whole koi herpes virus, trapped within a liposomal compartment ), replication-defective viruses, and attenuated replication-competent viruses (as summarized by Koelle, D.M. and L. Corey.
  • Attenuation of viruses can be spontaneous or can be induced by drugs (mutagenic or other nature such as for example UV light; se e.g. Mutation Research 768, 2016, 53-67 and J. gen. Virol, 1985, 66, 2271 -2277).
  • Mutations may relate to multiple viral mechanisms including replication capacity, spread of the virus, etcetera. Certain molecular pathways essential for virus replication and infectivity in vivo may be not essential for replication culture, and those genes involved in such pathways are likely more prone to genetic alterations during long-term culture.
  • mutations can be used to setup a vaccine approach with a discontinuously replicating virus.
  • the mutated virus is grown in a genetically engineered cell line that provides the required non-mutated gene in trans.
  • herpes simplex virus with a deletion of UL22 the late gene encoding gH, infects a non- complementing cell, the progeny virions can exit the cell but cannot infect a secondary cell (Koelle and Corey, 2003 as cited here above).
  • herpes virus genes of which dysfunction or deletion has been described to lead to functional attenuation of Koi Herpes Virus, an alloherpesvirus, or other herpesviruses. These genes are target genes for attenuation of the herpes virus according to the present invention:
  • TK thymidine kinase gene of Koi Herpes Virus.
  • a TK gene has also been described in Channel catfish herpesvirus (CCV), a virus relatively closely related to the virus described in the present invention (Hanson LA, Kousoulas KG, and RL Thune. 1994: Channel catfish herpesvirus (CCV) encodes a functional thymidine kinase gene: elucidation of a point mutation that confers resistance to Ara-T. Virology 202(2):659-64). 2) The d-UTPase gene of Koi Herpes Virus.
  • ORF57 of Koi Herpes Virus as given in Genbank accession N° NC_009127 where the ORF57 start and stop codon are located at position 99382 and 100803 (Boutier M, Ronsmans M, Ouyang P, Fournier G, Reschner A, et al. (2015): Rational Development of an Attenuated Recombinant Cyprinid Herpesvirus 3 Vaccine Using Prokaryotic Mutagenesis and In Vivo Bioluminescent Imaging. PLoS Pathog 1 1 (2): e1004690).
  • the gD (EHV en BHV) / gp50 (PRV) gene as found in bovine herpesvirus, equine herpesvirus en pseudorabiesvirus (Aujeszky's disease). This gene encodes a glycoprotein to which neutralizing antibodies can be generated.
  • the invention also pertains to a method of prophylactically a treating a an animal (i.e. for treating an animal to prevent a post treatment infection with the corresponding wild type pathogen) with a vaccine as described here above, comprising systemically
  • Systemically administering a vaccine means to administer the vaccine such that it reaches the circulatory system of the body
  • Systemic administration can be performed e.g. by administering the antigens into muscle tissue (intramuscular), into the dermis (intradermal), underneath the skin (subcutaneous), underneath the mucosa (submucosal), in the veins (intravenous), into the body cavity (intraperitoneal) etc.
  • the invention is also embodied in an antibody or antiserum reactive with a virus according to the invention and in a diagnostic test kit for the detection of antibodies reactive with a virus according to the invention or with antigenic material thereof, wherein the test kit comprises a virus according to the invention or antigenic material thereof.
  • the invention is also embodied in a diagnostic test kit for the detection of a herpesvirus according to the invention or antigenic material thereof, wherein said test kit comprises antibodies reactive with a virus according to the invention or with antigenic material thereof or a PCR primer set as described here above.
  • Example 1 Discovery of Lates Calcarifer Herpes Virus Collection of serum and tissue samples for isolation of an infectious agent
  • Scale drop disease virus which has localized, patchy lesions that are more deeply affected by necrosis with severe scale loss. Scale drop disease virus also typically causes a more chronic outbreak. Based on the observed difference, and the fact that a Scale drop disease virus PCR gave negative results, presence of a different infectious viral agent was suspected based on clinical observations on the affected farm. It was decided to do a follow up study aimed at virus isolation, and a new virus was found. Other observations on the diseased fish, apart from scale loss, were acute episodes of lethargy, severe inappetence, cloudy/swollen eyes and high mortalities (up to 30-70% of affected fish). Samples (serum, kidneys, spleens) were taken from affected fish. Pooled serum samples were stored at -70°C until further analysis. Pooled kidney samples were kept at +4°C until homogenization the next day.
  • SBB Seabass brain
  • V51 1/SBB_4P of the CPE-causing agent were analyzed using VIDISCA-454 technology described by De Vries et al. (201 1 ) PLoS ONE 6(1 ):e161 18. In both types of samples, sequences were obtained that were suspected to originate from a novel fish pathogen. These sequences were used to derive PCR primers for conventional PCR and quantitative PCR (see Table 2a-e). Blasting of the new sequences revealed that the CPE-causing agent in the culture supernatant and the suspected infectious agent in serum show a certain level of homology to viruses of the family of Alloherpesviridae. This new virus is henceforward called Lates Calcarifer Herpes Virus (LCHV).
  • LCHV Lates Calcarifer Herpes Virus
  • the virus culture supernatant sample was centrifuged for 10 minutes at 10,000 x g and treated with TurboDNase (Thermofisher) as described (de Vries M, et al. PLoS One. 201 1 ;6(1 ):e161 18. doi:10.1371/journal. pone.00161 18), after which nucleic acids were extracted by Boom extraction method (Boom R, et al. J Clin Microbiol. 1990;28(3):495- 503). The samples were sheared using dsDNA Fragmentase (New England Biolabs).
  • the sheared samples were purified with AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) in a ratio 1 :1.8 (sample:beads) to remove the enzymes. After purification the samples were end repaired with DNA polymerase I, Large (Klenow) Fragment (New England Biolabs). The end repaired samples were purified with AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) in a ratio 1 :1 .8 (sample:beads) to remove the enzymes, after which the samples were A-tailed by using Klenow Fragment (3'D5' Exo-) (New England Biolabs).
  • the samples were purified with AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) in a ratio 1 :1 .8 (sample:beads) to remove the polymerases.
  • Bubble adaptors from the NEBNext Multiplex Oligos for lllumina (New England Biolabs) were ligated to the A-tailed samples, by use of T4 ligase (Thermofisher).
  • a size selection was performed by use of AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) first in a ratio 1 :0.5 (sample:beads) to ensure that most fragments with a size bigger than 400 bp were removed, followed by adding additional AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) to the supernatant to get to a final ratio of 1 :0.85 (sample:beads) to bind DNA fragments between 200-400 bp and to remove fragments smaller than 200 bp.
  • Bubble adaptors were opened by using USER enzyme from the NEBNext Multiplex Oligos for lllumina (New England Biolabs).
  • a 28 cycle PCR was performed with adaptor specific primers from the NEBNext Multiplex Oligos for lllumina (New England Biolabs)and Q5 hotstart mastermix (New England Biolabs); 30 sec 98°C, and cycles of 10 sec 98°C and 75 sec 65°C, followed by 5 min 65°C.
  • the samples underwent size selections by use of AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) in a ratio 1 :0.5 (sample:beads) to remove fragments with a size bigger than 400 bp, and to the supernatant additional AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) were added to get to a final ratio of 1 :0.85 (sample:beads) to bind DNA fragments between 200-400 bp and to remove fragments smaller than 200 bp.
  • the concentration of the DNA was measured via Qubit dsDNA HS Assay Kit (Thermofisher), the size was checked on the bioanalyzer with a High Sensitivity DNA Analysis Kit.
  • the DNA was diluted to a concentration of 2.49 ng/ ⁇ . They were sequenced by use of the MiSeq (lllumina) using paired end sequencing and the v2 kit (lllumina).
  • PCR primers were designed on a 208 bp DNA fragment of the Lates Calcarifer Herpes Virus that was discovered in samples of the disease outbreak. This fragment shows homology at the translated nucleotide level with ORF62 of lctalurid Herpes virus 1 NP_041 153.2. Primers were also designed on the MEP, dUTPase, Terminase and Polymerase genes.
  • a master mix was made containing 1 x Supertaq buffer, 0.02 U/ ⁇ Supertaq enzyme, 0.2 mM deoxyribose nucleoside triphosphates (dNTPs), 1 ⁇ forward and 1 ⁇ reversed primer.
  • dNTPs deoxyribose nucleoside triphosphates
  • For each sample 2.0 ⁇ DNA template was added in 48 ⁇ PCR mix, 2 L sterile water was used as negative control.
  • a PCR program was designed starting with 60 seconds initialization at 95°C, followed by 40 repeats of denaturation, annealing and elongation for 30 seconds each at respectively 95°C, the primer set- specific annealing temperature based on Tm of the primers, and 72°C. The program ended with a final elongation at 72°C for 10 minutes. Samples were loaded with 1 x ethidium bromide in 1 .5% agarose gel and 1 x TAE buffer at 1 15 Volt for 60 minutes.
  • Quantitative Polymerase Chain Reactions were performed using a BioRad CFX 96 system. qPCR experiments were performed using Probe Fast Master Mix together with a sequence-specific probe. Each reaction existed of 18 ⁇ master mix containing 1 x Probe Fast q-PCR Master Mix (KAPA), 200 nM forward primer, 200 nM reversed primer and 200 nM probe. Primer pair 2 was used for qPCR analysis, with an optimized Tm of 60.7°C.
  • the probe DNA sequence was CGCGGGATGACCTCTTCTCG (SEQ I D NO:31 ) and the labels used were 5' 6FAM and 3' TAMRA. To 18 ⁇ of the master mix, 2 ⁇ DNA template was added.
  • a dilution series containing a pUC57 vector with Lates Calcarifer Herpes Virus-identity- sequence construct (synthesized by GenScript [the 208 bp fragment as mentioned above was synthesized and cloned in a plasmid vector]) was used as positive control, indicator for efficiency and accuracy and for sample quantification.
  • the vector was dissolved and diluted in water in a range of 1 .0 x10 1 copies / 2 ⁇ _ to 1.0 x 10 9 copies / 2 ⁇ _ per qPCR reaction.
  • the dilution series were included in all qPCR experiments and stored at -20°C.
  • Example 3 Experimental infection of the novel infectious agent in Asian Seabass Experimental infection of V511/SBB 4P in Asian Seabass (Lates calcarifer)
  • Table 3 Treatment groups and tank allocation.
  • V51 1/SBB_4P The viral titer of the CSF was 3.2 x 10 6 TCID 50 /mL (see below for titration protocol).
  • a second group of 25 fish were cohabitated in the same tank separated by netting (Table 3). The netting allowed free movement of water between the two tank halves and allowed close proximity, but not direct contact between fish of the two treatment groups. At the time of the start of the experiment, the fish averaged a weight of 18 grams.
  • Table 4 Sampling of fish tissue in experimentally IP infected group.
  • Table 7 Daily mortality and percentage cumulative mortality record*.
  • V51 1/SBB_4P virus supernatant transmitted from IP challenged to naive cohabitant fish Both groups experienced similar clinical symptoms. The clinical symptoms were also similar to those observed at the index farm (initial outbreak). The IP infected group experienced more acute and severe disease compared to the cohabitation group, with disease signs appearing within 3 days post-infection. In comparison, cohabitation challenged fish were showing initial clinical signs on day 7 post-infection.
  • Example 4 Sample preparation, tissue homogenization, DNA isolation of tissue samples collected during the infection experiment (Example 3)
  • DNA extraction was performed using a MagNA Pure 96 System and a MagNA Pure 96 DNA and Viral NA Kit. For extraction, 250 ⁇ MagNA Pure 96 External Lysis Buffer was added to 200 ⁇ sample. DNA was isolated with a pre-installed external lysis protocol and eluted in 50 ⁇ Milli-Q water. DNA was stored at -20°C until further use.
  • Example 5 Culture of the virus and titration of the virus
  • SBB Seabass Brain
  • the cell line SBB was originally derived at Intervet Norbio Singapore Pte Ltd (part of MSD AH) from a trypsinized suspension of Asian Seabass brain cells. Procedures for derivation of Seabass Brains cells have been described by Hasoon et al., in In Vitro Cell. Dev. Biol. - Animal 47: 16-25 (201 1 ) and by Chi et al. Dis Aquat Organ. 2005 Jun;65(2):91 -8.
  • the SBB culture medium consists of 899 ml E-MEM supplemented with 2 mM L- glutamine and 1 10 mg/L sodium pyruvate, 100 ml FCS (10%) and (optional) 1 mL of a Neomycin Polymyxin antibiotics solution 1000x stock. Cells were routinely grown at 28°C and 5% C0 2 .
  • the culture medium was kept at 4°C prior to startup of a culture.
  • One ampoule of frozen stock SBB was used to start a culture. Cells from liquid nitrogen were thawed fast by warming ampoule in 28°C water. The cell suspension was added to a tube and diluted slowly with 9 volumes of culture medium. Subsequently, cells were counted. The suspension was dispensed to the appropriate culture flask or roller bottle and incubated at 28°C and 5% C0 2 . Seeding density in the flask or roller bottle was approximately 3 x 10 4 cells / cm 2 . After 6-24 hours or complete attachment of the cells, the culture medium was refreshed to remove the remaining DMSO (freeze medium consists of 90% culture medium and 10% DMSO). The cells were further incubated for 3-7 days or until confluence was reached. For roller bottles, a roller speed of 0.2-0.5 rpm was required. Roller bottles can be of different surface areas of 480, 960 and 1750 cm 2 .
  • cells were passaged.
  • the passage can be performed every 3-4 days with an initial seeding density of 3.0 x 10 4 cells / cm 2 . Alternatively the passage can happen every 7 days when plated at a density of 1.0 x 10 4 cells/cm 2 .
  • the reagents for cell passage (medium, PBS, Trypsin/EDTA were pre-warmed to 28°C. The culture medium was discarded and the confluent monolayer was washed once with an appropriate volume of PBS (3 mL for a T25 flask).
  • the PBS was subsequently discarded and the cells were incubated in an identical volume of PBS supplemented with 1 % (vol/vol) of a 2.5% trypsin solution and 1 % (vol/vol) of a 2% EDTA solution for 15 minutes at 28°C. After detachment, an identical volume of fresh culture medium was added and the cells were resuspended and counted. A new flask was set up at the desired cell density in a culture volume appropriate for the culture flask or roller bottle.
  • culture medium and 2x concentrated freeze medium 80% (vol/vol) culture medium plus 20% (vol/vol) DMSO were kept at 4°C prior to the procedures.
  • the confluent cell culture was treated as described above up to and including trypsinization.
  • Cells were resuspended, counted, further resuspended in a suitable amount of culture medium, and an equal volume of 2 x freeze medium was added drop by drop while swirling the suspension.
  • Ampoules for liquid nitrogen storage were filled with 5.25 x 10 6 cells per ampoule to start a T175 or with 2.25 ⁇ 10 6 cells to start a T75.
  • the cells cultured from liquid nitrogen storage were passaged at least once before inoculation experiments were set up. The cells were passaged and cultured 24 hours before inoculation at
  • tissue culture flasks 3.0 x 10 4 cells/cm 2 in tissue culture flasks.
  • the inoculum consisted a fresh or freeze- thawed culture undiluted supernatant from the previous passage of the virus. The culture medium was removed from the flask. The flask was subsequently inoculated at 28°C for at least 60 minutes.
  • a MOI of 0.001 -0.01 TCID 50 per cell is used.
  • DNA for (quantitative) PCR was isolated from tissue culture medium using the MagNA Pure 96 System and a MagNA Pure 96 DNA and Viral NA Kit (Example 4).
  • SBB cells were cultured as described above. On the day prior to the test, a SBB cell suspension containing 6.0 x 10 4 cells/mL in culture medium (EMEM + 10% FCS + L-Glu + NaPyr) was prepared. The 96 wells of a microtiter plate were seeded with 100 ⁇ _ of this cell suspension. The plates were incubated for 24 hours at 28°C and 5% C0 2 . The monolayer was about 50% confluent after this incubation period.
  • culture medium EMEM + 10% FCS + L-Glu + NaPyr
  • TCID 50 was determined according to the method and calculations described by Reed and Muench, am. J. Epidemiol. (1938) 27(3): 493-497. qPCR analysis of DNA samples isolated from positive wells in the titration assay confirmed presence and replication of the virus.
  • Tissue culture flasks were seeded at 3.0 x 10 4 cells/cm 2 and cultured 24 hours. After 24 hours, cells in one flask were counted after trypsinization to determine the actual number of cells present in the flask. The culture medium was removed from the other flasks that were subsequently inoculated. Inoculum at 0.001 TCID 50 per cell consisting of an undiluted culture supernatant from a previous passage of LCHV virus in culture medium (passage number of the virus between 4-8) was applied to the monolayers and incubated for 60 minutes. The inoculum was removed and fresh culture medium was added to the cell culture flask. Cells were cultured until full CPE was observed using an inverted light microscope.
  • Virus was harvested by collection of the culture supernatant which was spun 5 min at 800 x g to remove debris. Samples were taken from (1 ) the undiluted inoculum used for infection of the monolayer, (2) the culture supernatant of a flask harvested 1 hour after replacing the inoculum with fresh medium, (3) a monolayer at 50% CPE and (4) a monolayer at 100% CPE. Titrations were performed on samples (1 ), (3) and (4), and qPCR analyses were performed on samples (1 ), (2), (3) and (4). The results are presented in Table 8. Pictures were captured at a 40 x magnification using an Olympus CKX41 inverted light microscope. These are shown in figure 2. Figure 2A shows morphology of SBB cells (p18) at 90% confluence and figure 2B at 50% CPE in a culturing flask. Table 8: Detailed results LCHV growth on SBB cells.
  • Copper grids of 400 mesh with a pure carbon film were exposed for 20 sec to a glow discharge in air to make the film surface hydrophilic.
  • Virus samples was placed on the carbon-coated grid with a volume of 10 ⁇ and left to incubate for approximately 2 min. Excess sample was blotted with a filter paper and 10 ⁇ of water was placed on the grid and immediately removed again by blotting. Then 10 ⁇ I of 1 % uranyl acetate was placed on the grid for staining. After 30 seconds, excess stain was removed by blotting and the specimen was left to dry for a few minutes before viewing in the electron microscope. Specimens were observed in a JEOL 101 1 transmission electron microscope operating at 80 kV. Images were recorded using a SIS Veleta 2kx2k camera.
  • Panel B shows a magnification in which the icosahedral contour of this particle can clearly be observed.
  • No enveloped viruses were found in the sample.
  • Figure 3 shows the LCHV virus captured with an Electron Microscope. In figure 3A two Herpes viruses (dark spots) are recognizable. The scale bar is 500 nm.
  • Figure 3B shows a magnification of one of the spots, clearly showing an icosahedral contour.
  • the examples presented above describe detection and isolation of a novel pathogen from diseased fish. The identical disease symptoms could be reproduced after experimental infection of healthy fish with the isolated pathogen. The infectious agent isolated from the experimentally infected fish was identical to the pathogen that was initially isolated. This is proof that the disease symptoms described above are solely attributable to the pathogen that was discovered, Lates Calcarifer Herpes Virus.
  • Example 7 primary cell culture Seabass fin cells
  • a primary cell culture from seabass fin cells (SBF) was established. Cells were cultured for at least 5 passages in culture.
  • the culture was established as follows. Fish were anesthetized. The caudal fin (tail) was trimmed and washed three times in PBS + gentamicin 0.3% + enrofloxacin 0.002% + amphotericin 0.5%. The fins were cut into tissue fragments using a scalpel blade. The fragments were transferred into 25 cm2 tissue culture flasks containing L15 culture medium supplemented with 20% FCS and gentamicin 0.3% + enrofloxacin 0.002% + amphotericin 0.5%. The flask was incubated at 28°C in a humidified incubator without C02. The medium was changed (L15) as required based on presence of debris and pH.
  • Cells were passaged by trypsinisation with 0.125% trypsin in PBS till cell detachment and split at low ratio between 1 : 1 -3, depending on cell density. During the initial passages cells were cultured in L15 medium supplemented with 20%FCS. The percentage FCS was reduced to 10% in later passages. Inoculation with Lates Calcarifer Herpes virus was carried out using procedures as described in Example 5. Day 4 post infection 100% CPE was observed.
  • Example 8 Additional strain of the novel virus
  • Terminase PCR fragment was sequenced and showed 97% identity with the sequence of Lates Calcarifer Herpes Virus as presented in SEQ ID NO:1 .
  • SEQ ID NO:29 presents the PCR product of the Terminase PCR of LCHV as presented in SEQ ID NO:1.
  • SEQ ID NO:30 presents the PCR product of the Terminase PCR of LCHV obtained from the farm different from the index farm. It was concluded that the outbreak of disease was caused by another strain of LCHV.

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Abstract

La présente invention concerne un nouveau virus pathogène de poisson provoquant une maladie chez le poisson, appelée virus de l'herpès de Lates calcarifer (LCHV), des cultures cellulaires comprenant ledit virus, des fragments d'ADN et des protéines correspondantes dudit virus, des vaccins à base dudit virus, un ADN et/ou une protéine et des anticorps réagissant avec ledit virus et des kits de test de diagnostic pour la détection dudit virus.
PCT/EP2017/070336 2016-08-11 2017-08-10 Nouveau virus pathogène de poisson WO2018029301A1 (fr)

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CN110564750A (zh) * 2019-08-30 2019-12-13 中国水产科学研究院长江水产研究所 一种鲫造血器官坏死症酵母口服疫苗及应用
CN113462694A (zh) * 2021-05-24 2021-10-01 广西科学院 一种针对大口黑鲈虹彩病毒感染细胞的核酸适配体及其应用
CN113462694B (zh) * 2021-05-24 2024-04-05 广西科学院 一种针对大口黑鲈虹彩病毒感染细胞的核酸适配体及其应用
CN116267799A (zh) * 2022-12-30 2023-06-23 中国科学院水生生物研究所 一种抗鲫疱疹病毒银鲫的创制方法
CN116267799B (zh) * 2022-12-30 2024-04-09 中国科学院水生生物研究所 一种抗鲫疱疹病毒银鲫的创制方法

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