DK180123B1 - Salmon Gill Poxvirus - Google Patents

Abstract

The present document is directed to a new poxvirus infecting salmon. The present document further discloses the genomic sequence of this double-stranded DNA virus and the use of this sequence information for detection, diagnosis and/or vaccine development for the virus.

Description

PISCINE POXVIRUS - METHODS AND MEANS FOR DETECTION AND DIAGNOSIS

TECHNICAL FIELD

The present document discloses a virus, herein denoted Piscine poxvirus. The present document is further directed to methods and means for detection and diagnosis of said virus infection as well as products fro use in the prevention and/or treatment of said virus.

BACKGROUND

Poxviruses differ from other viruses in their size and complexity. They are large, with linear, double stranded DNA encoding for about 200 proteins and is the only known DNA virus that replicates entirely in the cytoplasm¹. They are reported in both invertebrates (Entomopoxvirinae) and vertebrates (Chordopoxvirinae). Poxvirus infections, in general, are acute, with no strong evidence for latent, persistent, or chronic infections². Variola virus, the causative agent of smallpox, belongs to this group of virus. The vaccinia virus used to prevent smallpox. The narrow host specificity of Variola virus was important in the eradication of smallpox³.

Hence, some members of the Poxviridae can cause severe, systemic disease, while others, can cause mild, localized disease depending on the virus species, the route of entry, the host species and its immune status⁴ The host tropism varies from narrow to broad⁵ and the virus is epitheliotrophic, typically causing proliferation of epithelial cells⁶.

In general, little is known about poxvirus infections in aquatic animals^7-10.

SUMMARY

The present document discloses a virus herein denoted Piscine poxvirus or alternatively salmon gill poxvirus (SGPV) (both used interchangeably herein), characterized in that it comprises a nucleic acid sequence according to SEQ ID NO:1 or a nucleic acid sequence having at least 85% identity thereto, and/or a nucleic acid sequence complementary thereto. The present document is also discloses a Piscine poxvirus, wherein the genome of said Piscine poxvirus consists of a nucleic acid sequence according to SEQ ID NO:1 or a nucleic acid sequence having at least 85% identity thereto, and a nucleic acid sequence complementary thereto.

The present document is also directed to an isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence according to any one of SEQ ID NO:1-9 or a variant thereof having at least 85% identity thereto, or a nucleic acid sequence complementary thereto. Further disclosed herein is a nucleic acid fragment of said isolated nucleic acid molecule, said fragment comprising or consisting of at least 5 contiguous nucleic acid bases of a nucleic acid molecule according to any one of SEQ ID NO:1-9. The nucleic acid fragment may be a nucleic acid primer or nucleic acid probe capable of specifically detecting Piscine poxvirus in a sample, said probe optionally further comprising one or more label(s) for detection of said probe. Such a nucleic acid probe may comprise at least 5, such as about 5-300, 5-100, 10-100, 15-80, 15-50, 18-35, or 15-25 contiguous nucleotides of a nucleic acid sequence according to any one of SEQ ID NO:1-3 or a sequence having at least 85% identity thereto, or a sequence complementary thereto, and optionally one or more label(s) for detection of said probe. The nucleic acid sequence of a nucleic acid probe may be a nucleic acid sequence of any one of SEQ ID NO:4-7. Suitable labels include, but are not limited to, a fluorescent label, such as TAMRA (tetramethylrhodamine), FAM 6-carboxyfluorescein), NED^TM, VIC®, and/or HEX^TM. A fluorescent label may be used together with MGBNFQ (Minor Groove Binding NonFluorescence Quencher).

The present document is also directed to an isolated nucleic acid fragment as defined herein, wherein said nucleic acid fragment is a primer consisting of at least 5, such as about 5-50, 10-40, 18-30, 12-35 or 15-28 contiguous nucleotides of a nucleic acid sequence according to any one of SEQ ID NO:1-9, such as SEQ ID NO:1-3, or a sequence complementary thereto. A primer may be as defined in any one of SEQ ID NO:8-9.

The present document is also directed to a vector comprising one or more isolated nucleic acid molecule(s) and/or nucleic acid fragment(s) as defined herein. Also disclosed is a host cell comprising one or more nucleic acid molecule(s) and/or nucleic acid fragment(s) or a vector as defined herein.

The present document is further directed to a polypeptide encoded by a consecutive string of at least 12 nucleic acid bases of an isolated nucleic acid molecule or fragment as defined herein, or a nucleic acid sequence reverse complementary thereto. Exemplary polypeptides include, but are not limited to, the peptides according to any one of SEQ ID NO:10-15. Also disclosed herein is an antigen comprising or consisting of a polypeptide as defined herein. Further disclosed is an antibody specifically directed to such an antigen.

The present document is also directed to the use of a nucleic acid fragment as defined herein and/or an antibody as defined herein for detecting the presence (or absence) of a virus, such as specifically detecting the presence or absence of Piscine poxvirus (and/or Piscine poxvirus specific nucleic acids or peptides/proteins) and/or diagnosing a viral infection, such as Piscine poxvirus infection, in a sample. For example, in situ hybridization or polymerase chain reaction may be used for detecting the presence or absence of the Piscine poxvirus and/or diagnosing the Piscine poxvirus viral infection.

The present document is also directed to the use of an isolated nucleic acid molecule or a nucleic acid fragment as defined herein, such as a nucleic acid according to any one of SEQ ID NO:1-9, for preparing a primer and/or probe which specifically detects Piscine poxvirus. Such a primer and/or probe may be used for analysing the presence or absence of Piscine Pox virus in a sample, such as a tissue sample from fish. Such a primer and/or probe is further disclosed elsewhere herein.

The present document is also directed to the use of an isolated nucleic acid molecule or a nucleic acid fragment as defined herein for expressing a peptide, or the use of a polypeptide as defined herein, for preparing an antibody which specifically detects Piscine Pox virus. Such an antibody may be used for analysing the presence or absence of Piscine Pox virus in a sample, such as a tissue sample from fish. Such an antibody is further disclosed elsewhere herein.

The present document is also directed to a method for detecting a virus, such as Piscine poxvirus, said method comprising detecting at least 5 consecutive nucleic acid bases of a nucleic acid sequence according to any one of SEQ ID NO:1-9, or a sequence having at least 85% identity thereto, or a sequence complementary thereto. Such a method may comprise performing a polymerase chain reaction or in situ hybridisation.

The present document is further directed to a method, such as an ex vivo method for detecting the presence of a Piscine poxvirus specific nucleic acid, Piscine poxvirus specific polypeptide or protein, and/or Piscine poxvirus and/or diagnosing a Piscine poxvirus infection in a sample, said method comprising the steps of:

a) contacting the sample with a nucleic acid fragment as defined herein, and/or an antibody as defined herein;

b) detecting the formation of a complex between a Piscine Poxvirus specific nucleic acid or polypeptide, respectively, and said nucleic acid fragment or antibody, respectively.

wherein the presence of a complex indicates the presence of a Piscine poxvirus specific nucleic acid, Piscine poxvirus specific polypeptide or protein, and or Piscine Poxvirus and/or a Piscine Poxvirus infection in said sample.

The method may be performed ex vivo.

In all aspects of the present document a sample includes, but is not limited to, a tissue sample from fish, such as salmon, rainbow trout, or carpe. The sample may e.g. be a tissue sample from gills, pseudobranc, blood, heart, liver, kidney, spleen, pancreas, pylorus or skeletal musculature, central nervous system, in particular gills.

The present document is also directed to a diagnostic kit for diagnosing a viral infection, such as Piscine poxvirus infection, in a subject, said kit comprising one or more of an isolated nucleic acid molecule as defined herein, a nucleic acid fragment as defined herein, a vector as defined herein, a host cell as defined herein, a polypeptide as defined herein, an antigen as defined herein, and/or an antibody as defined herein and reagents for performing a diagnosis, and optionally instructions for performing such a diagnosis.

The present document is also directed to an isolated nucleic acid molecule as defined herein, a nucleic acid fragment as defined herein, a vector as defined herein, a host cell as defined herein, a polypeptide as defined herein, an antigen as defined herein and/or an antibody as defined herein for medical use.

The present document is also directed to a pharmaceutical composition comprising an isolated nucleic acid molecule as defined herein, a nucleic acid fragment as defined herein, a vector as defined herein, a host cell as defined herein, a polypeptide as defined herein, an antigen as defined herein, and/or an antibody as defined herein. Also disclosed is such a pharmaceutical composition for use as a vaccine.

The present document is also directed to an isolated nucleic acid molecule as defined herein, a nucleic acid fragment as defined herein, a vector as defined herein, a host cell as defined herein, a polypeptide as defined herein, an antigen as defined herein, an antibody as defined herein and/or a pharmaceutical composition as defined herein for use for the prevention and/or treatment of a Piscine poxvirus infection.

The present document is also directed to an isolated nucleic acid molecule as defined herein, a nucleic acid fragment as defined herein, a vector as defined herein, a host cell as defined herein, a polypeptide as defined herein, an antigen as defined herein and/or an antibody as defined herein for use in the preparation of a medicament for the prevention and/or treatment a Piscine poxvirus infection.

The present document is also directed to the use of an isolated nucleic acid molecule as defined herein, a nucleic acid fragment as defined herein, a vector as defined herein, a host cell as defined herein, a polypeptide as defined herein, an antigen as defined herein and/or an antibody as defined herein for the preparation of a medicament for the prevention and/or treatment a Piscine poxvirus infection.

The administration of a substance or pharmaceutical composition of the present document may e.g. take place by intraperitoneal injection, dip vaccination, bath vaccination and/or by oral vaccination.

The present document is also directed to the use of a nucleic acid molecule as defined herein as a vector and a vector comprising at least part of an isolated nucleic acid molecule according to any one of SEQ ID NO: 1-9, such as SEQ ID NO 1-3, or a variant thereof.

Other features and advantages of the invention will be apparent from the following detailed description, drawings, examples, and from the claims.

DEFINITIONS

As used herein, the term “nucleic acid sequence”, “nucleic acid molecule”, “nucleic acid” and the like refers to a polynucleotide molecule (DNA - deoxyribonucleic acid, or RNA ribonucleic acid) comprising a string of nucleic acid bases. These nucleic acid bases are “A” (adenine), “T” (thymidine)/“U” (uracil), “C” (cytidine) and “G” (guanidine). In RNA, “T” is replaced with “U”. DNA or RNA may be single-stranded or double-stranded. By an RNA sequence “corresponding to” a nucleic acid sequence expressed as a DNA sequence, the same nucleic acid sequence but wherein “T” is replaced by “U” to get the corresponding RNA sequence is intended. The term “nucleic acid” may comprise both DNA and/or RNA sequences unless one or the other is specifically referred to. Preferably, nucleic acid sequences are DNA sequences in the present document.

cDNA (complementary DNA) can be produced by reverse transcription of RNA.

As used herein in connection with nucleic acid molecules (DNA and RNA molecules) and polypeptides, the term “isolated” means that the molecule or polypeptide has been removed from its original environment. This means that a nucleic acid molecule or polypeptide when present in a living organism is not “isolated” whereas the breaking of chemical bonds and/or by other means separating the sequence from its natural environment (such as by isolating it from the virus particle) means that the nucleic acid molecule or polypeptide is “isolated”.

By “identity” is in the context of the present document intended the extent to which two (nucleic or amino acid) sequences have the same residues at the same positions in an alignment, expressed as a percentage. A local algorithm program may be used to determine sequence identity. Local algorithm programs, (such as Smith Waterman) compare a subsequence in one sequence with a subsequence in a second sequence, and find the combination of subsequences and the alignment of those subsequences, which yields the highest overall similarity score. Internal gaps, if allowed, are penalized. Local algorithms work well for comparing two multidomain proteins, which have a single domain or just a binding site in common. Methods to determine identity and similarity are codified in publicly available programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J et al. (1984)) BLASTP, BLASTN, and FASTA¹¹ (Altschul, S.F. et al. (1990)). The BLASTX program is publicly available from NCBI and other sources 11(BLAST Manual, Altschul, S.F. et al. (1990)). Each sequence analysis program has a default scoring matrix and default gap penalties. In general, a molecular biologist would be expected to use the default settings established by the software program used. By e.g. a sequence having 95 % identity it is intended that the amino acid or nucleotide sequence is identical to the reference sequence, except that the amino acid/nucleotide sequence may include up to 5 point mutations per each 100 amino acids or nucleotides of the reference amino acid/nucleotide sequence. In other words, to obtain an amino acid/nucleotide sequence having at least 95% identity to a reference sequence up to 5% of the amino acids/nucleotides in the reference sequence may be deleted or substituted with another amino acid/nucleotide, or a number of amino acids/nucleotides up to 5% of the total number of amino acids/nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the terminal positions of the reference amino acid or nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids or nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

By “variant”, “variant thereof” or “variants thereof” as used in the present document is intended a nucleic acid or polypeptide sequence(s) having an identity to a specified nucleic acid or polypeptide sequence of at least 85% or at least 90%, such as 85-100%, 86-100%, 87-100%, 89-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95100%, 96-100%, 97-100%, 98-100%, 99-100% or about 100%. Unless something else is explicitly mentioned herein, whenever a nucleic acid or fragment or part thereof is referred to in this document, also intended is a nucleic acid having at least 85% identity, such as 85-100%, 86-100%, 87-100%, 88-100%, 89-100%, 90-100%, 91-100%, 92-100%, 93100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%, 99-100% or about 100% identity, such as about 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto. Such a nucleic acid fragment may encode a polypeptide having substantially the same biological activity as the referred to nucleic acid would.

A “probe”, such as a DNA probe, refers to an isolated nucleic acid sequence capable of hybridizing to an, at least partially, complementary nucleic acid sequence. A probe often contains a label allowing detection of the complex formed between the probe and the target nucleic acid sequence. Examples of such probes include, but are not limited to radioactive probes, fluorescent agents, chemiluminescent agents, enzyme substrates and enzymes. Further information regarding the use and choice of labels can e.g. be found in ¹²(Sambrook et al. Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) and ¹³(Ausubel et al. Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-Intersciences (1987)).

A double stranded nucleic acid molecule (DNA or RNA molecule) consists of two “complementary” nucleic acid strands. Generally, “A” (adenine) is complementary to “T” (thymidine) in a DNA molecule and “U” (uracil) in an RNA molecule, while “C” (cytidine) is complementary to “G” (guanidine). A thus binds to T and G to C via hydrogen bonds. If in a double-stranded DNA molecule one strand reads “5'-ACGCT-3” its “complementary” strand reads “'3'-TGCGA'-5”. As used herein, the terms complement complementarity, complementary and the like, are thus used to describe single-stranded polynucleotides related by the rules of antiparallel base-pairing. A “reverse complementary” strand and the like expressions refers to a DNA sequence read in the reverse direction on the opposite strand, e.g. the reverse complementary strain to the sequence 5’-ATGC-3’ is 5’-GCAT-3’. Complementarity may be partial where the base pairing is less than 100%, or it may be complete or total, implying perfect 100% antiparallel complementation between the two polynucleotides. By convention in the art, single-stranded nucleic acid molecules are written with their 5' ends to the left, and their 3' ends to the right.

A “primer” is a nucleic acid strand which serves as a starting point for DNA synthesis. In DNA synthesis, the enzyme (DNA polymerase) used for catalysing DNA synthesis requires an existing nucleic acid strand in order to be able to add new nucleotides to an existing DNA strand. The primer is constructed so that it binds to another DNA strand by antiparallell base paring. In the PCR reaction, the primer is allowed to hybridize to a target DNA molecule whereafter the DNA polymerase synthesises new DNA using the target DNA molecule’s sequence as a template.

A “vector” is a DNA or RNA molecule used to carry foreign material to a cell. A vector typically contains sequences for its replication in a host cell and one or more transgene(s). It may also contain one or more promoter sequence for the expression of inserted genes and/or sequence regulating transcription and/or translation. Vectors are typically inserted into their target cells (host cells) by transformation (for bacterial cells, transfection (for eukaryotic cells) or transduction (often used terminology when a viral vector is inserted into a host cell). Viral vectors generally have a modified viral DNA or RNA rendering them non-infectious.

As used herein, a host cell includes an individual cell or cell culture which can be or has been a recipient of any vector of this document. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected with a vector comprising a nucleic acid of the present document. Host cells may be prokaryotic or eukaryotic cells.

By “polypeptide” is herein intended a string of amino acid bases linked by a covalent peptide (amide) bond between the carboxyl group of one amino acid to an amino group on the adjacent amino acid. Amino acid sequences are usually expressed with their Nterminal to the left and the carboxy-terminal on the right. A polypeptide is generally shorter than a “protein” which latter term is usually used for polypeptides being longer than 50 amino acids. Herein, these two terms may be used interchangeably independently of the length of the amino acid string.

The term antigen, as used herein, refers to any agent that is recognized by an antibody, while the term immunogen refers to any agent that can elicit an immunological response in a subject. The terms antigen and immunogen both encompass, but are not limited to, polypeptides. In most, but not all cases, antigens are also immunogens.

The term “antibody” is directed to an immunoglobulin molecule and immunologically active parts (fragments) of such immunoglobulin molecules. An antibody is capable of binding an antigen. Natural antibodies are Y-shaped protein molecules containing two each of a heavy chain and a light chain connected with each other by disulfide bonds. Although the overall structure of different antibodies is very similar, the tip of the antibody is highly variable allowing different antibodies to recognize different kinds of antigens. Antibodies are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. Antibodies may be coupled to labels, such as fluorescent, chemiluminescent or enzymatic labels, which allow their use for detection of certain antigens in situ and ex situ and diagnosis of disease.

A vaccine or a vaccine composition, as mentioned herein, is intended to refer to a composition resulting in immunological prophylaxis in a subject to which the vaccine is administered. A vaccine composition induces an immune response and thus a long-acting immunity, to a specific antigen. In the present context an antigen is mainly intended to refer to an inactivated form of a virus, or parts or fragments thereof which are still capable of generating an immune response in a subject.

PCR (polymerase chain reaction) is a method for amplification of nucleic acid molecules. The PCR reaction is well-known to the person skilled in the art and involves contacting a sample with a pair of so called oligonucleotide primers (one forward and one reverse primer) under conditions allowing the hybridization between the primers and a target (template) sequence having complementarity to the primers and which target sequence possibly is present in the sample in order to amplify the target sequence.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1: Phylogenetic analysis of Piscine poxvirus. The analysis was done using maximum likelihood (General Time Reversible substitution model) and rifampicin resistance protein sequences. Bootstrap values above 50% have been indicated.

Figure 2: IHC Piscine poxvirus (positive area indicated by arrow).

Figure 3: Normal tissues and pathology in SGPV-infected Atlantic salmon. (a) A normal gill with thin lamellae (arrows) ensures efficient gas exchange. Chloride cells are present in normal numbers and at the normal location (arrowheads). (b and c) Detaching apoptotic cells with central clearing of chromatin (arrows) in the nuclear seen by H&E staining (b) and confirmed by red TUNEL staining (c). (d and e) IHC staining of poxvirus (brown) as cytoplasmic granules (d) and apical budding processes from apoptotic gill epithelial cells (e). (f) H&E staining of collapsed, adherent (arrows) thin lamellae losing apoptotic epithelial cells, creating an atelectasis-like condition hindering gas exchange. (g) H&E staining of proliferating (the arrow indicates metaphase), pale, foamy epithelial cells occluding the normally water-filled interlamellar space for gas exchange. Chloride cells are displaced and degenerated (arrowhead). (h) The lesion in panel g stained by IHC for PCNA showing brown nuclei, including proliferating cells in metaphase (arrow). (i andj) The lesion in panel gstained by IHCfor chloride cells that are displaced and enlarged (i) compared to the chloride cells in a normal gill (j). (k) TEM showing virus particles consistent with poxvirus in size and shape. Note the presence of crescents (CR), immature virions (IVs), and mature virions (MVs). (l) H&E staining of prominent hemophagocytosis (arrows) in the hematopoietic interrenal tissue. Methods included H&E staining (a, b, f, g, l); IHC staining for TUNEL (c), salmon gill poxvirus (d, e), PCNA (h), and chloride cells (i, j); and TEM (k).

Figure 4: Distribution of SGPV genes by tiers of inferred origin. The number of genes in each tier and the percentage of the total are indicated. “NCLDV”, genes inferred to have been present in the common ancestor of all NCLDV; “poxvirus”, genes that originated in the common ancestor of the poxviruses; and “chordopoxvirus”, genes that originated in the common ancestor of chordopoxviruses; TM/SP, transmembrane helix/signal peptide.

Figure 5: Phylogenetic tree of poxviruses. The tree was constructed from a multiplesequence alignment of 13 proteins that are conserved in all poxviruses and ASFV (NCVOG0022, major capsid protein; NCVOG0023, D5-like heli-case-primase;

NCVOG0031, unclassified DEAD/SNF2-like helicases; NCVOG0038, DNA polymerase elongation subunit family B; NCVOG0076, DNA or RNA helicases of superfamily II; NCVOG0249, packaging ATPase; NCVOG0261, poxvirus early transcription factor [VETF], large subunit; NCVOG0262, poxvirus late transcription factor VLTF-3-like; NCVOG0267, RNA helicase DExH-NPH-II; NCVOG0271, DNA-directed RNA polymerase subunit beta; NCVOG0274, DNA-directed RNA polymerase subunit alpha; NCVOG1117, mRNA capping enzyme; NCVOG1164, A1L transcription factor VLTF-2). The root position was forced between the two families. Numbers at internal nodes indicate bootstrap support (on a scale of from 0 to 1).

Figure 6: Reconstruction of the evolution of the gene repertoire of the NCLDVs. The numbers at internal branches (shown only for the ASFV-Poxviridae branch and for the root) indicate the maximum likelihood estimates of the number of genes mapped to the respective ancestral form. The numbers after the virus names indicate the number of annotated genes. The NCLDV families used as outgroups are shown by triangles. The NCLDV tree topology is from reference 39.

Figure 7: Dot plot comparison of poxvirus gene orders. Each dot corresponds to a pair of orthologous genes. The horizontal axis shows the SGPV genes, and the vertical axis shows the GenInfo Identifier sequence identification numbers for genes of the respective viruses.

Figure 8: Alignment of the genome architectures of SGPV and VACV. The alignment was generated using the Artemis tool and the table of gene orthology derived from the NCVOG assignments obtained in this work. The orthologous genes are connected by red lines, and the names of the respective vaccinia virus genes are indicated. nt, nucleotides.

Figure 9: Synteny-based evolutionary tree of poxviruses. The root between chordopoxviruses and entomopoxviruses was forced. The tree was constructed using the neighbor-joining method, and the distances between the genome architectures of the respective viruses that were estimated as described previously (48) are shown in the table underneath the tree; a unit distance means that the fraction of orthologous gene pairs that belong to synteny blocks is equal to e—1. Amsmo, Amsacta moorei entomopoxvirus; Melsa, Melanoplus sanguinipes entomopoxvirus; Vacco, vaccinia virus; Deevi, deerpox virus; Psevi, pseudocowpox virus; Canvi, canarypox virus; Crovi, crocodilepox virus; Squvi, squirrelpox virus; Molco, molluscum contagiosum virus; Yabvi, Yaba-like disease virus.

Figure 10: Phylogenetic tree of the viral B22R-like genes. The numbers on the left show bootstrap values as percentages. The bar shows the scale as the estimated number of amino acid substitutions per site. For the cyprinid herpesviruses, the GI numbers are indicated on the right. The three paralogs from SGPV are shown in red. The chordopoxvirus sequences are collapsed and shown as a triangle.

DETAILED DECRIPTION

The present document discloses a poxvirus infecting fish, herein denoted Piscine poxvirus or alternatively salmon gill poxvirus (SGPV) (Fig. 2). Both Piscine poxvirus and salmon gill poxvirus may be used herein to denote this virus. The inventors recognize that this is the first poxvirus identified by molecular methods that infects fish. The virus causes severe disease with massive mortalities in farmed Atlantic salmon. The infection has a wide geographical distribution in Norway, affects all age groups of fish and preliminary results suggest no infection of other salmonids. The samples of the virus used for sequencing according to the present document was obtained from salmon from the northern part of Norway. Preliminary results demonstrate that the Piscine poxvirus infects primarily the gills and causes extensive gill pathology with severe respiratory disease. Pathological changes are also seen in kidney and spleen. However, these symptoms may not be directly linked to the virus itself, but may be due to some kind of pathophysiological consequence of the infection.

In 2012, the inventors collected material from an Atlantic salmon fresh water farm experiencing acute high mortality with respiratory disease suspected to be the primary problem. In addition to autopsy and histological examination of several organs, the inventors investigated the gills from two diseased salmons by transmission electron microscopy (TEM). Based on these results, a specimen was selected for high throughput sequencing (for further information see the experimental section). Total RNA was isolated and sequenced. The sequencing yielded 521 710 reads. All reads were translated into protein sequences (all reading frames and both strands) and sequence similarity searches against all available poxvirus sequences available from GenBank using tblastx (Altschul et al. (1990) Basic local alignment search tool. J. Mol. Biol. 215:403-410) were performed. Two reads with weak similarity to known poxvirus sequences were identified and one of those reads was used to design a real-time PCR assay. Using the assay, a sample with high viral DNA content was selected for Illumina sequencing.

Based on analyses of the genomic sequence prepared, a large number of potential open reading frames (genes) could be identified. When using the megablast algorithm with default parameters and nucleotide data, surprisingly, no matching sequence regions were found in National Center for Biotechnology Information’s complete collection of viral sequences. Even when looking at potential protein sequences, very few viral matches exceed 4-5 consecutive amino acids. Phylogenetically, the virus appears to belong to a new group of poxviruses (Figure 1).

Due to the substantial lack of homologous sequences from other Poxviruses, the sequencing of the novel piscine Poxvirus was not straightforward. However, due to this uniqueness, most loci are suitable for the design of both specific probes and primers for e.g. detection/diagnosis of the virus, without risking any substantial cross-reaction to other Poxviruses (or other viruses). Also, due to the genome's uniqueness most loci are suitable for the development of recombinant vaccines. Also, as the genome is unique, so are the peptides and proteins which can be expressed based on the genome's sequence. This is relevant for the specificity of detection methods based on detection of proteins/peptides and vaccines based on in vivo or in vitro expressed proteins/peptides.

The present document is directed to an isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence according to SEQ ID NO:1-9 or a variant thereof having at least 85% identity thereto, such as 85-100%, 86-100%, 87-100%, 88-100%, 89100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%, 99-100% or about 100% identity thereto, or a nucleic acid sequence complementary to said nucleic acid sequence or variant thereof.

The present document is also directed to an isolated nucleic acid molecule, wherein said nucleic acid molecule is a fragment of a nucleic acid sequence according to SEQ ID NO:1-9, such as SEQ ID NO:1-3, wherein said nucleic acid fragment comprises or consists of at least 5 contiguous nucleic acid bases, such as about 5-1000, 5-900, 5-800, 5-700, 5-600, 5-500, 5-400, 5-300, 5-200 or 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 530, 5-25, 5-20, 10-100, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 15-100, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, 15-25, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 17-50, 1835, 15-25, 20-30, 18-35 or 15-25 contiguous nucleic acid bases of a nucleic acid sequence according to SEQ ID NO:1-9, or a variant thereof, or a nucleic acid sequence complementary to said nucleic acid sequence according to SEQ ID NO:1-9 or variant thereof.

As mentioned above, the Piscine poxvirus genome turned out to be very unique with little sequence similarity to other poxviruses (or other viruses generally). Most parts of the genome will therefore be unique to the Piscine poxvirus and thus suitable for the development of primers, probes, peptides etc. specific for the Piscine poxvirus. For example, the region of SEQ ID NO:1 spanning from nucleotides 1-5679 (SEQ ID NO:2), the sequence of which is also duplicated between nucleotides 235 885-241 564, is very unique to the Piscine poxvirus. Another region which is very unique to the Piscine poxvirus is the region of SEQ ID NO:1 spanning from nucleotides 99 129 to 99 230 (SEQ ID NO:3). The sequence according to any one of SEQ ID NO:1-9, such as SEQ ID NO:1, 2 and/or 3 may thus be used for preparing a primer and/or probe for specifically detecting Piscine poxvirus (i.e. the primer and/or probe specifically detects Piscine poxvirus) and/or for diagnosing a Piscine poxvirus infection. Such a primer and/or probe may hybridize to a Piscine poxvirus nucleic acid sequence under high stringency conditions.

The sequence according to any one of SEQ ID NO:1-9, such as SEQ ID NO: 1,2 and/or 3 may also be used for expressing a polypeptide as defined herein for preparing an antibody which specifically detects Piscine poxvirus. The peptides disclosed herein according to SEQ ID NO:10-15 may also be used for preparing an antibody which specifically detects Piscine Poxvirus. Such a primer, probe and/or antibody may be used for analysing the presence or absence of Piscine poxvirus in a sample, such as a tissue sample from fish. Examples of samples to be analysed are disclosed elsewhere herein and include tissue samples form fish and water samples.

A fragment of a nucleic acid as disclosed herein may be a nucleic acid primer or nucleic acid probe capable of detecting or specifically detects Piscine poxvirus in a sample. Such a primer or probe is thus capable of specifically detecting Piscine poxvirus in a sample. By “specifically detecting” or “specifically detects” it is intended that the primer or probe allows the detection of Piscine poxvirus without any cross-reactivity with other known piscine viruses. Preferably, the primer and/or probe does not show any cross-reactivity with other poxviruses either. Cross-reactivity with other poxviruses is not a major concern when working with samples from fish, as Piscine poxvirus is the only poxvirus identified from fish. Most preferably, the primer/probe does not show any cross-reactivity with any other nucleic acid sequences. The primers/probes therefore preferably hybridizes specifically to a Piscine poxvirus nucleic acid sequence as disclosed herein without hybridisation to other nucleic acids. A primer/probe is considered to hybridize specifically when it hybridizes solely to a nucleic acid from Piscine poxvirus. Preferably said hybridization is performed under high stringency conditions.

The expressions “specifically detecting” or “specifically detects” may also be used in the context of immunological (immunohistochemical) detection methods and/or antibodies for detecting Piscine Pox virus. In this context it is intended that the immunological detection method or antibody allows the detection of Piscine poxvirus without any cross-reactivity with other known piscine viruses. Preferably, the antibody does not show any crossreactivity with other poxviruses either. Most preferably, the antibody does not show any cross-reactivity with any other protein. A possible cross-reactivity with other poxviruses is not a major concern when analysing the presence of Piscine poxvirus in tissue samples from fish, as Piscine poxvirus is the only known poxvirus in fish. The antibody thus specifically binds to a peptide or protein from Piscine poxvirus without showing any crossreactivity to any other piscine viruses or peptides or proteins generally.

The present document is therefore also directed to a nucleic acid probe comprising at least 5, such as about 5-300, 5-200 or 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 525, 5-20, 10-100, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 15-100, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, 15-25, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 17-50, 18-35, 1525, 20-30, 18-35 or 15-25 contiguous nucleotides of a nucleic acid sequence according to SEQ ID NO:1-9, such as SEQ ID NO:1-3, or a variant thereof, or a sequence complementary thereto. The nucleic acid part of the probe may consist of a nucleic acid sequence as described above, even if the probe also may include other parts, such as one or more label(s) for detection of said probe or said probe when bound to a target nucleic acid. A probe is generally at least 15 nucleic acid bases long, such as about 1535, such as 15-35, nucleic acids long, but it may be shorter. The skilled person knows how to select a suitable length for a probe depending on its use. The nucleic acid part of a nucleic acid probe may e.g. comprise or consist of the nucleic acid sequence of SEQ ID NO:4-7.

A primer or a probe in accordance with the present document may be constructed having high specificity, i.e. a large sequence identity, to a target sequence to allow for a specific detection of a target sequence, or it may be constructed with a lower sequence identity to a target sequence to allow for detection of a target sequence having a lower sequence identity to the probe. The specificity of a primer or probe may be affected by the length of the primer or probe. Primer or probe specificity may also be affected by the conditions used for hybridization, such as salt concentration, temperature and pH. A person skilled in the art knows how to elaborate with these so called stringency conditions used for hybridization to affect primer-target or probe-target sequence complex formation. By increasing the pH and/ temperature and/or lowering the salt (sodium ion) concentration, the hybridization conditions provide for a higher stringency (i.e. high stringency conditions), i.e. rendering the formation between a primer or a probe and a target sequence more difficult. The person skilled in the art also knows how to elaborate with sequence specificity to a target sequence in order to obtain a primer and/or probe having the desired sequence specificity.

A probe according to the present document generally contains one or more labels which allow the detection of the probe and optionally one or more label(s) for detection of said probe. Examples of such labels include, but are not limited to radioactive labels, fluorescent agents, chemiluminescent agents, enzyme substrates and enzymes. Further information regarding the use and choice of labels can e.g. be found in Sambrook et al. Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al. Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-Intersciences (1987). A radioactive label may e.g. be ¹⁴C, ³²P, ³⁵S, ³H or ¹⁵O, which may be detected using suitable radiation detection means. A fluorescent label may e.g. be a fluorescent dye, such as rhodamine, SYBR Green, fluorescein, thiazole orange, FAM, FAM 6-carboxyfluorescein, fluorescein istothiocyanate (FITC), or TAMRA (tetramethylrhodamine), NED^TM, VIC®, and/or HEX™. Such probes are well known to the person skilled in the art and may be commercially obtained.

MGBNFQ (minor groove binding non-fluorescence quencher) may also be used in combination with a fluorophore to increase the specificity of a shorter probe. MGB itself is not fluorescent. Dihydrocyclopyrroloindole tripeptide minor groove binder (MGB) is a modification of the probe that increases the specificity of probe binding to template DNA. The most commonly used quencher in MGB probes is Non-Fluorescence Quencher, hence the long acronym MGBNFQ. The most commonly used reporter dye is 6-FAM (6carboxyfluorescein), but a whole array of other fluorophore may be used as dyes/quenchers, including TAMRA (tetramethylrhodamine), NEDTM, VIC®, and/or Hex^tm. 6-FAM may for instance be used as a reporter dye in combination with TAMRA as a quencher, or HEX^TM.may be used as a reporter dye with TAMRA as a quencher.

The label may also be a chemiluminescent agent, an enzyme substrate and/or an enzyme, such as ß-galactosidase, horseradish peroxidase, streptavidin, biotin or digoxigenin.

Examples of methods where probes may be used include, but are not limited to, in situ hybridization, analysis of nucleic acid fragments on gels, real time PCR, digital PCR etc.

Methods where probes are used generally involve denaturing double-stranded nucleic acids (i.e. separating the two nucleic acid strands from each other) in a sample, allowing the probe to bind, wash off any unbound probe and detecting the formation of a probetarget sequence complex. However, during real time PCR, the probe instead binds to the target sequence, and is then fragmented by Taq polymerase during the elongation step. Thus, no washing off of the unbound probe is necessary in real time PCR. Methods involving the use of probes include, but are not limited to, polymerase chain reaction (PCR) and in situ hybridisation.

The isolated nucleic acid molecules disclosed herein or variants thereof may also be used for the construction of primers for a PCR reaction. The person skilled in the art is well acquainted with how such primers are to be prepared. The present document is therefore also directed to a nucleic acid fragment, wherein said nucleic acid fragment is a primer consisting of at least 5, such as about 5-50, 10-40, 18-30, 12-35 or 15-28 contiguous nucleotides of a nucleic acid sequence according to SEQ ID NO:1-9, or a variant thereof, or a sequence complementary thereto. A primer having a length of about 18-30 bases is generally considered to ensure an adequate specificity of the primer while the primer is still sufficiently short to easily bind to the template nucleic acid. However, a primer in accordance with the present document may be both shorter and longer. The skilled person knows how to select a suitable length for a primer depending on its use. Exemplary primers are disclosed in SEQ ID NO:8-9. A forward and a reverse primer for use in a real-time PCR reaction for specifically detecting piscine Poxvirus are typically separated by 250 base pairs or less, preferably 200 base pairs or less, although they may be separated by a higher number of base pairs.

For exemplary purposes only, a PCR based detection of Piscine poxvirus may utilize SEQ ID NO:8 as a forward primer, SEQ ID NO:9 as a reverse primer and a probe comprising SEQ ID NO:4 and MGB and a fluorescent probe, such as the ones specified above. Any example of a probe labelled with a reporter dye and a quencher will work. One example is an MGB-modified probe with NFQ quencher (‘MGBNFQ’) and 6-FAM as reporter dye.

One region of the Piscine poxvirus genome that is suitable for constructing specific primers, probes, polypeptides and/or antibodies is the region spanning from nucleotide 1 to nucleotide 5679 in SEQ ID NO:1, herein denoted SEQ ID NO:2. This is a sequence that is very distinct for the Piscine poxvirus but that is likely to be less variable on a population level. The region is duplicated and also present at nucleotide position 235 885 to 241 564 of SEQ ID NO:1.

PCR (polymerase chain reaction) is a method for amplification of nucleic acid molecules, well-known to the person skilled in the art. In short, PCR involves contacting a sample with a pair of so called oligonucleotide primers (one forward and one reverse primer) under conditions allowing the hybridization between the primers and a target (template) nucleic acid sequence having complementarity to the primers (i.e. the formation of a complex between the respective primers and the target sequence). The target (template) nucleic acid may e.g. be Piscine poxvirus DNA. The primers are constructed to bind on the 3' side of the sense and antisense strands of the target sequence, respectively. Thereafter the primers are extended by using a polymerase, dissociated from the template, re-annealed, extended, dissociated in a number of cycles. The number of cycles may be adjusted depending on the amount of target sequenced present in the sample and the amount of copies needed but is typically 20-40 although it may be both higher and lower. If the target sequence was present in the sample, the PCR reaction will allow for the provision of a number of copies of it (the amplification product).

A PCR reaction may e.g. be used to amplify a nucleic acid sequence e.g. for its subsequent use in a cloning reaction wherein the amplified nucleic acid sequence is inserted into another nucleic acid molecule, such as a vector, or for its sequencing. A PCR reaction may also be used for analyzing a sample for the presence of a specific target sequence, as amplification of the sequence will only occur if the target sequence is present in the sample. The amplification product can be analyzed e.g. by electrophoresis, probe hybridization and/or sequencing.

PCR may also be made quantitative, so that the initial amount of a target nucleic acid in a sample, and consequently e.g. a virus containing this target sequence, can be quantified. PCR can be made quantitative (qPCR) and allow for real time measurement of the amplified product by the use of fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan.

The present document is also directed to a vector comprising one or more nucleic acid molecule(s) and/or nucleic acid fragment(s) as defined herein.

The Piscine poxvirus, or parts thereof, may also be used as a viral vector for inserting foreign material in a cell. For this purpose, one or more isolated part(s) or the whole of the Piscine poxvirus genome as defined in SEQ ID NO:1, or a DNA sequence complementary thereto, may be used, such as an isolated nucleic acid molecule according to any one of SEQ ID NO:1-9, such as SEQ ID NO:1-3. The present document is thus also directed to a vector comprising one or more isolated part(s) or the whole of the Piscine poxvirus genome as defined in SEQ ID NO:1, or a DNA sequence complementary thereto, such as an isolated nucleic acid molecule according to any one of SEQ ID NO:1-9, such as SEQ ID NO:1-3 or a variant thereof. Also, it is possible to recombinantly assemble different parts of the isolated Piscine poxvirus genome to construct a recombinant vector. Importantly, such a vector should not contain any infectiously harmful sequence(s). The vector may be used for carrying foreign material into a cell by inserting genetic material of interest into the vector. To accomplish this, the vector is preferably constructed to contain one or more multiple cloning sites allowing for specific opening of the vector to insert the genetic material of interest. The vector may also be constructed to contain sequences for expression of inserted genetic material, such as promoter sequences, ribosome binding sites and/or sequences regulating the translation of the inserted genetic material. Viral vectors generally have a modified viral DNA or RNA rendering them non-infectious.

The present document is also directed to a host cell comprising one or more nucleic acid molecule(s) or fragment(s) thereof, or variant(s) thereof, as disclosed herein or a vector as disclosed herein. The host cell may be a prokaryotic or eukaryotic host cell. A host cell typically allows for the amplification and/or replication of the genetic material inserted (e.g. nucleic acid(s) and/or vector(s)) therein.

The isolated nucleic acid molecules, and fragments and variants thereof may also be used for expressing polypeptides. The present document is therefore also directed to a polypeptide encoded by a consecutive string of at least 12 nucleic acid bases, such as about 12-1000, 12-900, 12-800, 100-500, 100-400, 150-400, 150-300, 12-100, 12-90, 1280, 12-70, 12-60, 12-50, 12-48, 50-800, 50-700, 50-600, 50-500 nucleic acid bases of a nucleic acid molecule according to SEQ ID NO:1-9 or fragment or variant thereof as defined herein, or a nucleic acid sequence reverse complementary thereto. Such a polypeptide may e.g. be used as an antigen for the preparation of an antibody and/or it may be used to elicit an immune response in an organism. When a variant of a nucleic acid molecule or a fragment of such a nucleic acid molecule as defined herein is used for producing a polypeptide in accordance with the present document, such a polypeptide may have substantially the same biological activity as a polypeptide encoded by a sequence identical to SEQ ID NO:1-9 or a fragment thereof. Exemplary polypeptides constructed based on the Piscine poxvirus genome are shown in SEQ ID NO:10-15.

Also disclosed herein is an antigen comprising a polypeptide as defined herein. Such an antigen may be used for the preparation of an antibody capable of binding specifically to said antigen. Such an antibody may e.g. be used for the detection of a Piscine poxvirus specific peptide in a sample. The antibody may in addition or alternatively also neutralize or reduce the function or activity the antigen (polypeptide). The present document is therefore also directed to an antibody specifically directed to an antigen as defined herein. The antibody may be e.g. a polyclonal antibody or a monoclonal antibody. The antibody may e.g. be a teleost antibody or a chimeric antibody. An antibody for specificially detecting Piscine poxvirus in a sample, such as a tissue sample from fish, may be directed to a surface protein of said Piscine poxvirus.

An antigen may also be used for the preparation of a vaccine composition used for eliciting an immune response to such an antigen. Such a vaccine composition may be used for the prevention and/or treatment of a Piscine poxvirus infection.

The nucleic acid molecule(s) disclosed in the present document or fragments thereof or variants thereof may be used as primer(s) or probe(s), e.g. for specifically detecting the presence of a Piscine poxvirus specific nucleic acid in a sample and/or for diagnosing a Piscine poxvirus infection in a subject. In particular, in situ hybridization or polymerase chain reaction may be used for such detection and/or diagnosis. Also, an antibody as disclosed herein may be used for specifically detecting the presence of Piscine poxvirus in a sample and/or diagnosing a Piscine poxvirus infection in a subject. Such a detection of an antibody may be effected by labelling the antibody with a label, such as an enzymatic or fluorescent label. Commonly used labels for antibodies include, but are not limited to horseradish peroxidase, alkaline phosphatase and biotin.

Studies of mammalian animal models have demonstrated that protection against disease is associated with antibody responses to both infectious intracellular/mature virion (MV) and extracellular form (EV) of vaccinia virus, which is another poxvirus infecting mammals. The present virus, Piscine poxvirus, has been found to be very distant from other poxviruses in its nucleic acid sequence, i.e. there is a low sequence homology between Piscine poxvirus and other poxviruses. However, even if it is difficult to identify homologues of poxvirus proteins based on e.g. structural similarities, proteins that have been important for the development of vaccines against other poxvirusesmay be identified. Examples of vaccinia proteins which are expected to have a functional homologue in Piscine poxvirus and which may be of interest as targets for detection of

Piscine poxvirusus and/or in vaccine development include for example the proteins of Table 1. These proteins have been shown to be targets for neutralizing antibodies for mammal.

Table 1. Examples of vaccinia proteins which are expected to have a functional 5 homologue in Piscine poxvirus

Protein	Location	Role	Neutralize	Ref
A27	MV	Attachment	MV	Ref 14
A28	MV	Entry / Fusion	MV	Ref 17
D8	MV	Attachment	MV	Ref 18
H3	MV	Attachment	MV	Ref 20
L1	MV	Entry / Fusion	MV	Ref 25
A33	EV	Spread	-	Ref 22
B5	EV	Spread	EV	Ref 22

The L1 protein is reported to be conserved throughout the poxvirus family and is nearly identical in vaccinia and variolavirus and is an important component in current experimental vaccines²⁵ (Su 2005). The present inventors have generated antibodies 10 against the corresponding protein in Piscine poxvirus. This was primarily done using amino acid sequence similarity searches, the L1 protein from Piscine poxvirus can for instance be aligned over 66% of the total length and shows 27 % sequence identity (vaccinia virus vs. Piscine poxvirus) in the aligned region when using protein BLAST. Antibodies were generated to the sequences L1 amino acids 221-235 (SEQ ID NO:10) 15 and L1 amino acids 2-20 (SEQ ID NO:11). An antibody was also generated to the whole L1 protein (SEQ ID NO:12).

Antibodies have also been generated against protein P4 of Piscine poxvirus (major core protein, SEQ ID NO: 15). Antibodies were generated against P4 amino acids 214-233 (SEQ ID NO:13) and P4 amino acids 453-470 (SEQ ID NO:14). An antibody was also 20 generated to the full P4 protein.

In situ hybridization involves detecting a specific DNA or RNA nucleic acid in a sample, such as a tissue sample. The method generally comprise the steps of fixating the sample, allowing a probe to hybridize to complementary DNA or RNA in the sample, washing off unbound probe and thereafter detecting the formation of a complex between the probe and a target nucleic acid possibly present in the sample. A probe for use in an in situ hybridization reaction is preferably longer than a probe used for detection of a PCR amplified nucleic acid fragment, and is typically about 35 nucleic acid bases long. For exemplary purposes only, in situ hybridization based detection of Piscine poxvirus may involve a probe according to any one of SEQ ID NO:5-7.

The present document is also directed to a method for detecting Piscine poxvirus, said method comprising detecting at least 5, at least 7, at least 10, at least 12, at least 15, such as 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 15-100, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, or 20-40, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleic acid bases of a nucleic acid sequence according to any one of SEQ ID NO:1-9, or a variant thereof, or a sequence complementary thereto.

Methods suitable for use in such a method include, but are not limited to, polymerase chain reaction (PCR) or in situ hybridisation (both being disclosed in further detail elsewhere herein).

The present document is also directed to a method for detecting the presence of a Piscine poxvirus specific nucleic acid, a Piscine poxvirus specific polypeptide or protein, and/or Piscine poxvirus and/or diagnosing a Piscine poxvirus infection in a sample, said method comprising the steps of:

a) contacting the sample with a nucleic acid fragment as defined herein, a probe as defined herein, and/or an antibody as defined herein;

b) detecting the formation of a complex between a Piscine poxvirus specific nucleic acid or polypeptide, respectively, and said nucleic acid fragment or probe, or antibody, respectively.

wherein the presence of a complex indicates the presence of a Piscine poxvirus specific nucleic acid, a Piscine poxvirus specific polypeptide or protein, and/or Piscine poxvirus and/or a Piscine pox viral infection in said sample. The sample may contain Piscine poxvirus specific nucleic acids, such as DNA, and/or Piscine poxvirus specific polypeptides/proteins.

The method may be performed ex vivo. In an ex vivo method, the biological sample is first isolated from the organism to be tested before the analyzing the presence or absence of

Piscine poxvirus. The ex vivo method may be e.g. PCR or in situ hybridization. In situ hybridization may involve the use of nucleic acid based probes binding to Piscine poxvirus specific nucleic acids. The ex vivo method may also be an immunohistochemical method using antibodies binding to Piscine poxvirus specific peptides and/or proteins. The sample to be tested for the presence of Piscine poxvirus may also be a non-biological sample, such as a water sample.

The method for for detecting the presence of Piscine poxvirus and/or diagnosing a Piscine poxvirus infection may be followed by administering a pharmaceutical composition, such as a pharmaceutical composition as disclosed herein, in order to treat the Piscine poxvirus infection.

In all aspects of the present document, a sample to be analysed for the presence of Piscine poxvirus or a Piscine poxvirus specific nucleic acid or polypeptide may be a biological sample, such as a tissue sample from fish, such as salmon, carpe or rainbow trout. The tissue sample may be a tissue sample from e.g. gills, pseudobranc, blood, heart, liver, kidney, spleen, pancreas, pylorus or skeletal musculature, or the central nervous system, in particular gills.

The present document is also directed to a diagnostic kit for detecting the presence or absence of Piscine poxvirus in a sample, such as for diagnosing a Piscine poxvirus infection in a subject,said kit comprising one or more of a nucleic acid molecule as defined herein, a nucleic acid fragment as defined herein, a vector as defined herein, a host cell as defined herein, a polypeptide as defined herein, an antigen as defined herein, and/or an antibody as defined herein and reagents for performing a diagnosis, and optionally instructions for performing such a diagnosis.

The present document is also directed to a nucleic acid molecule or variant thereof, a nucleic acid fragment or variant thereof, a vector, a host cell, a polypeptide, an antigen and/or an antibody for medical use.

The present document is also directed to a pharmaceutical composition comprising a nucleic acid molecule or variant thereof, a nucleic acid fragment or variant thereof, a vector, a host cell, a polypeptide, an antigen, and/or an antibody as defined herein. Such a pharmaceutical composition may be used as a medicine, such as a vaccine composition, e.g. for the prevention and/or treatment of a Piscine poxvirus infection.

As the Piscine poxvirus genome has a very low identity to other piscine viruses polypeptide sequences obtainable from the genome will represent proteins with unique features when compared with data in publicly available databases. In fact, the virus is so distant from other poxviruses that it is impossible to determine which is the closest relative. The most similar protein when comparing all proteins from Piscine poxvirus with all proteins from the vaccinia virus appears to be the DNA-dependent RNA polymerase subunit rpo132. Here, the blastx algorithm reports < 47% identity and the longest conserved sequence motif is ten amino acids.

The general idea of vaccinations is that the patient is exposed to a non virulent version of the pathogen, or parts of the pathogen (proteins) to which a protective response is generated. Alternatively, a synthetic vaccine (recombinant vaccine or DNA vaccine) may be produced instead by using the nucleic acid sequence information of Piscine poxvirus presented herein. By annotation (prediction of the biological function of the genes) of different sequences it is possible to choose the peptide sequences that most will trigger a protective immune response. A recombinant vaccine can for example be made by inserting the gene sequence for the proteins listed in Table 1 above into an expression system (e.g. E. coli or insect cells) to generate antigens. To generate a long term immune response the use of an adjuvant is recommended.

A pharmaceutical composition according to the present document may also comprise one or more adjuvant(s) (such as a mineral oil, muramyldipeptides, lipopolysaccharides, glucans and Carbopol®), pharmaceutically acceptable excipients, carrier(s), emulgator(s) etc. Liquid carriers include, but are not limited to water, petroleum, plant and animal oils, such as peanut oil, mineral oil, soybean oil, or sesame oil, and synthetic oils. A liquid composition may also comprise physiological saline solution, saccharide solutions (e.g. dextrose), glycols (e.g. ethylene glycol, propylene glycol, or polyethylene glycol. The active component of a pharmaceutical composition as disclosed herein may constitute about 0.5 to 90% by weight of the pharmaceutical composition. Methods and means for preparing a vaccine composition suitable for storage are well known for the skilled practitioner within this field.

Vaccine components may be in liquid form both as hydrophilic and lipophilic, which phased may often then be mixed in emulsions that need to be stabilized for storage. Examples of vaccine preparations suitable for vaccination of fish may be found in Roar

Gudding (Editor) et al. ”Fish Vaccinology”, Developments in Biological Standardization, 484 pages.

In addition, dry vaccines may also be prepared which are dissolved before use. Such vaccines are particularly useful for dip, bath or oral vaccines that are not using oil adjuvants or the like.

The present document is also directed to a nucleic acid molecule or variant thereof, a nucleic acid fragment or variant thereof, a vector, a host cell, a polypeptide, an antigen, an antibody and/or a pharmaceutical composition, as defined herein, for use for the prevention and/or treatment of a Piscine poxvirus infection.

Also disclosed herein is a nucleic acid molecule or variant thereof, a nucleic acid fragment of variant thereof, a vector, a host cell, a polypeptide, an antigen and/oran antibody , as defined herein, for use in the preparation of a medicament for the prevention and/or treatment a piscine poxvirus infection.

The administration may take place by intraperitoneal injection, dip vaccination, bath vaccination and/or by oral vaccination.

When a fragment as disclosed herein is used for medical purposes this typically does not contain any label.

The nucleic acids and polypeptides disclosed herein may be isolated.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXPERIMENTAL SECTION

Example 1: Molecular identification of the piscine poxvirus and elucidation of the complete genomic sequence

In 2012, the inventors collected material from an Atlantic salmon fresh water farm, experiencing acute high mortality with respiratory disease suspected to be the primary problem. In addition to autopsy and histological examination of several organs, the inventors investigated the gills from two diseased salmons by transmission electron microscopy (TEM). Based on histopathological observations, a high quality Atlantic salmon gill specimen that appeared to have a high viral load was identified. The material was of sufficient integrity that both DNA and RNA could be extracted for genetic characterization. The method used for identification of any infectious agent present was based on a strategy referred to as ‘computational subtraction', first published by Weber et al. (Nat Genet. 2002 Feb;30(2):141-2). Briefly, total RNA was reverse transcribed and amplified using the QuantiTect Whole Transcriptome Kit (QIAGEN). Total RNA was chosen as this would allow us to identify RNA viruses in addition to any DNA-based pathogen (DNA viruses, bacteria etc.). High throughput pyrosequencing was done and 521 710 reads were generated using a GS FLX Titanium sequencing machine (454 Life Sciences). Using the megablast algorithm ²⁷(Zhang et al. 2000. J. Comput. Biol., 7, 203214) for sequence comparison and the available sequence data from Atlantic salmon as a reference, all reads matching the salmon genome or transcriptome were removed. The remaining reads were assembled into contigs using the software Velvet assembler (Zerbino & Birney 2008. Genome Research 18 (5): 821-829) and a series of sequence comparison tools were used in an attempt to identify any infectious agents. No viruses, bacteria or protists could be found based on the nucleic acid sequence data or the inferred amino acid sequences. The inventors then went back the filtered sequences reads and did sensitive amino acid-based sequence similarity searches against databases of known viral sequences. When translating both the inventors' sequence database and the virus sequence database into all reading frames and including the reverse complement version of both sequence sets, three individual reads were found to have a weak similarity with proteins from known poxviruses. Based on one of these reads, a real-time PCR assays was designed. The primers/probe set (forward primer ATCCAAAATACGGAACATAAGCAAT (SEQ ID NO:8), reverse primer

CAACGACAAGGAGATCAACGC (SEQ ID NO:9), MGB probe

CTCAGAAACTTCAAAGGA (SEQ ID NO:4) - all written 5'-3') was used to assess viral load in several series of samples collected from suspected poxvirus outbreaks as well as healthy fish. There was a clear quantitative and qualitative correlation between PCR results and gill pathology. It thus concluded that the sequence did indeed stem from a causally involved virus. Using the real-time PCR assays a specimen was identified that appeared to have a particularly high amount of poxvirus nucleic acids. Poxviruses have DNA genomes, so in order to get more sequence information, total DNA was prepared and sequences using a paired-end strategy and the HiSeq 2500 System (Illumina). Paired-end sequencing will produce pairs of reads that stem from the same sequence regions. The library was designed using DNA fragments that had an average length of 370 basepairs and it was thus assumed that every pair of sequences (101 bases) in the database had this genomic distance between them.

The Illumina sequencing gave a total of 2 X 169 083 705 reads. All reads were assembled de novo into contigs using the Velvet assembler. A contig containing both original poxvirus reads was identified. This contig had flanking regions that appeared to be repetitive and the length (approximately 20 000 basepairs) indicated that this sequence fragment did not correspond to the complete poxvirus genome. Analyzing the contigs, reads from flanking regions where only one member of the paired reads was mapped could be identified. The rationale was that if one read maps to a particular contig and the corresponding paired read maps unambiguously to another contig, it is likely that these contigs stem from adjacent genomic regions. Primers were designed in order to link the original contig with what appeared to be flanking contigs and PCRs were performed from one contig to the next. This could be done in both directions and through multiple rounds of PCR and sequencing of PCR products, a 241 564 basepair contig could eventually be constructed. This supercontig did not appear to have any flanking contigs and it was concluded that this sequence represented the complete poxvirus genome.

Based on analyses of the genomic sequence, a large number of potential open reading frames (genes) could be identified. When using the megablast algorithm with default parameters and nucleotide data, no matching sequence regions were found in National Center for Biotechnology Information’s complete collection of viral sequences. Even when looking at potential protein sequences, very few viral matches exceed 4-5 consecutive amino acids.

Phylogenetically, the virus appears to belong to a new group of poxviruses (Figure 1). As mentioned above, due to the genome’s uniqueness most loci are suitable for the design of both specific probes and primers for e.g. detection/diagnosis of the virus. Also, due to the genome’s uniqueness most loci are suitable for the development of recombinant vaccines. Also, as the genome is unique, so are the peptides and proteins which can be expressed based on the genome sequence. This is relevant for the specificity of detection methods based on detection of proteins/peptides and vaccines based on in vivo or in vitro expressed proteins/peptides.

Example 2: Immunological detection of Piscine poxvirus

Sections from gills were dewaxed, rehydrated, treated with trypsin 1/100 at 37°C for 90 min for antigen demasking, washed and the reaction was stopped by incubating the sections in ice cold TBS for 15 min. The sections were incubated for 20 min in Trisbuffered saline (TBS 0.05 m, pH 7.6) with 5% bovine serum albumin (BSA) for prevention of non-specific binding, tilted to remove solution, incubated for at 4°C overnight with a rabbit antibody (Pacific Immunology) generated against the synthesized peptide of SEQ ID NO: 11 that is part of the L1 protein, a transmembrane protein expressed on the surface of the IMV. The antibody was diluted 1/5000 in 2.5 % BSA. Visualization was performed using EnVision Kit (Dako) with HRP and AEC as a chromogen or an alkaline phosphatase / fast red visualizing system. The same approach was performed to other antibodies as disclosed herein. Immunohistochemistry with the full protein antibodies will be tested as soon as the antibodies arrive.

Example 3: Isolation of DNA and PCR protocol

DNA was isolated from heart, kidney, liver, spleen, muscle and gill tissue. Approximately 20 mg of tissue was homogenised in Lysing Matrix D containers (MP Biomedicals GmbH) with 200 pl lysis/binding solution (MagMAX-96 Total RNA Isolation Kit, Ambion) and 1.4 pl β-mercaptoetanol by use of a rotor stator homogenizer. The RNA isolation kit was used according to the manufacturers’ recommendations. To perform the magnetic based separation, a KingFisher (Labsystems Oy) was used. After elution, the DNA concentration and purity was measured using a NanoDrop ND-1000 spectrophotometer (Nano - Drop Technologies). All samples had OD260/280 ratios between 1.8 and 2.2. Between 200 and 1000 ng DNA was added to the reaction. The Platinum Quantitative PCR SuperMix-UDG (Life Technologies) was used with primer concentrations of 500 nM, probe concentration 300 nM and the following PCR cycle: 2 min at 50°C (UDG incubation), 15 min at 95°C (inactivation of UDG), followed by 50 cycles of 15 seconds at 94°C (template denaturation), 30 seconds at 55°C (primer annealing) and 15 seconds primer elongation.

Detailed description of experiments

MATERIALS AND METHODS

Sample material. Samples were collected from three different Norwegian salmon farms in which the fish had suspected SGPV-related disease (gill apoptosis) (Table 2) and at the following clinical stages: premortality (n = 20; samples were taken 1 to 3 days before mortality was observed), mortality (n = 60; samples were taken from tanks in which mortality occurred and lethargic fish crowded on the bottom), and postmortality (n = 10; samples were taken from tanks in which mortality was observed a week prior to sampling). The average weight was 27 g (range, 10 to 40 g).

TABLE 2. Overview of material from Norwegian salmon farms

Cases (gill apoptosis)	Archival cases 19952006	Controls (no gill apoptosis)
Diseased	Farm A	Farm B	Farm C	Fish (n) / farms	Diseased fish	Healthy fish
fish:	fish(n)	fish(n)	fish(n)	(n)	(n) / farms (n)	(n) / farms (n)
Premortality	20	-	-
Mortality*	30	25	5	39 / 14	48 / 8	3 / 1
Postmortality	10	-	-

*Sampling was performed on 5 dead and 25 moribound fish. Except for the 5 dead fish, all fish used in this study were sampled while still alive.

Archived, formalin-fixed, paraffin-embedded (FFPE) gill tissue was identified from 14 cases with records of gill disease and apoptotic gill epithelial cells. These cases were geographically spread in both fresh- and seawater sites in Norway (Table 2). Included were 12 fish from the first known outbreak of so-called amoebic gill disease in Norway (32). A separate TEM study also demonstrated poxvirus-like particles in those 12 fish. In addition, samples from 48 fish with other gill diseases (without gill epithelial apoptosis) and 3 healthy fish were included as controls (Table 2; see also Table 4).

Tissue sampling for histology, TEM, and PCR. All fish were anesthetized and autopsied, and gill tissues were fixed in neutral phosphate-buffered 10% formalin for histology and in RNAlater (Qiagen Inc., Valencia, CA, USA) for quantitative PCR (qPCR). Additional organs sampled for histology were heart, liver, intestine, spleen, kidney, muscle, and skin. Additional organs sampled for PCR were spleen, kidney, and skin from five fish in the premortality stage and five dead fish from farm A (Table 2). Formalin-fixed gill tissue from one fish in the premortality stage and one fish in the mortality stage was prepared for TEM as described previously (33).

In situ staining methods. Paraffin-embedded and hematoxylin and eosin (H&E)-stained sections were made for histology. For a subset of the samples, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in situ cell death detection kit AP (Roche, Basel, Switzerland) was used to confirm apoptosis.

Prussian blue staining was used to verify hemosiderosis in the spleen and kidney. Staining for osmoregulatory chloride cells and proliferating cell nuclear antigen (PCNA) was performed as described previously (34, 35).

Immunohistochemistry (IHC) for SGPV. Sections from gills were dewaxed, rehydrated, treated to demask antigen, and blocked with 5% bovine serum albumin in Tris-buffered saline to prevent nonspecific binding. Sections were incubated at 4°C overnight with a rabbit antibody (Pacific Immunology, Ramona, CA, USA) generated against the synthesized peptide GVNVDVKEFMQKFESNLSN-Cys (SEQ ID NO:11) which is part of the L1 protein, a transmembrane protein expressed on the surface of the intracellular mature virion). Visualization was performed using an EnVision kit (Dako, Glostrup, Denmark) with horseradish peroxidase and 3-amino-9-ethylcarbazole as the chromogen.

DNA isolation and qPCR detection. DNA was isolated from various tissues using a QIAcube system and a QIAamp DNA minikit according to the manufacturer’s recommendations (Qiagen Nordic, Oslo, Norway). For archive material, a QIAamp DNA FFPE tissue kit was used (Qiagen Inc., Valencia, CA, USA). A qPCR assay based on the SGPV genomic sequence was designed for the molecular detection of virus DNA. The target locus was the homolog of the vaccinia virus (VACV) D13L open reading frame (ORF), which has been suggested to be a unique feature of poxviruses. The assay comprised forward primer ATCCAAAATACGGAACATAAGCAAT (SEQ ID NO:8), reverse primer CAACGACAAGGAGATCAACGC (SEQ ID NO:9), and the minor groove binding (MGB) probe CTCAGAAACTTCAAAGGA (SEQ ID NO:4) labeled with 6carboxyfluorescein and a minor groove binding nonfluorescence quencher (MGBNFQ). The assay was run using a Platinum quantitative PCR SuperMix-uracil DNA glycosylase (UDG) kit (Life Technologies AS, Oslo, Norway) and the following PCR parameters: 50°C for 2 min (for UDG incubation), 95°C for 15 min (for UDG inactivation), and 50 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 15 s. Reactions with threshold cycle (C_T) values above 40 were repeated for confirmation of the results. All results with C_T values above 45 were considered negative. In this study, there were no C_T values between 35 and 45.

When sections were cut from archival FFPE tissue, healthy fish gill tissue samples were interspersed between the samples from different disease outbreaks to control for carryover contamination. Isolation of DNA from FFPE tissue sections was done with a QIAamp DNA FFPE tissue kit according to the manufacturer’s instructions (Qiagen Inc., Valencia, CA, USA).

RNA and DNA sequencing. On the basis of the findings of TEM analyses, an Atlantic salmon gill tissue specimen containing poxvirus-like particles was selected for highthroughput sequencing. Total RNA was isolated from gill tissue fixed in RNAlater (Qiagen Norge, Oslo, Norway) using an RNeasy kit (Qiagen) and treated with Turbo DNA-free DNase (Life Technologies AS, Oslo, Norway) according to the manufacturer’s recommendations. After DNase inactivation, 50 ng of total RNA was reverse transcribed and amplified using a QuantiTect whole-transcriptome kit (Qiagen). An initial round of total RNA pyrosequencing was done using a Roche 454 GS-FLX system and Titanium chemistry (454 Life Sciences, a Roche Company, Branford, CT, USA). All reads (521,710) from all reading frames and both strands were translated into protein sequences, and searches for sequence similarity to all poxvirus sequences available in GenBank were performed using the tblastx program (11). Two reads with weak similarity to known poxvirus sequences were identified, and one of these reads was used to design a qPCR assay (forward primer, ATCCAAAATACGGAACATAAGCAAT (SEQ ID NO:8); reverse primer CAACGACAAGGAGATCAACGC (SEQ ID NO:9); MGB probe,

CTCAGAAACTTCAAAGGA (SEQ ID NO:4); all sequences are written 5' to 3'). Using the assay, a gill tissue sample with a high viral DNA content was selected for a subsequent round of sequencing. Total DNA was prepared using a DNeasy kit (Qiagen) and sequenced directly using a paired-end strategy and an Illumina HiSeq 2500 system (Illumina, Inc., San Diego, CA, USA). Reads were assembled de novo using a Velvet sequence assembler. Primers for PCR-based gap closing were designed using the software Primer Express (version 2.0.0; Applied Bio-systems, Life Technologies Corporation, Carlsbad, CA, USA), and PCR was performed using a HotStarTaq master mix kit (Qiagen). Amplification products were sequenced directly using Sanger sequencing.

The sequencing with the Illumina system gave a total of 169,083,705 pairs of 101-bp reads. Using the Velvet sequence assembler, a total of 68,968 high-confidence contigs could be generated (coverage, >10 times; length, >100 bp). A contig containing the two reads originally identified as being poxvirus-like was found, and pairs of reads where one partner mapped uniquely to one contig and the other mapped to a different contig were extracted using the poxvirus-like contig as a starting point. Using this information, 24 of the contigs produced by the Velvet sequence assembler could be arranged into a tentative scaffold of the genome. PCR was successful across all gaps, but for a small number of loci, the exact number of low-complexity repeats could not be established using

Sanger sequencing due to length and base compositional bias. Instead, the approximate lengths of repeat regions were determined using a 2100 Bioanalyzer and a DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA) to analyze the gap PCR products. Only 5 of the original 521,710 reads from the 454 sequencing data set could be mapped back to the final version of the virus genome. The five reads ranged in length from 52 to 528 bases, and the longest read was identical to the one that was used to design the PCR assay.

Genome annotation. The SGPV genome was translated by Gene-MarkS software (http://exon.biology.gatech.edu/); long (>80-nucleotide) intergenic regions were checked for the presence of ORFs, and ORFs ranging from 50 to 100 codons were annotated to be predicted protein-coding genes if they showed significant sequence similarity to other proteins or to a conserved domain in the National Center for Biotechnology Information Conserved Domains Database or contained predicted transmembrane helices and/or a signal peptide. Transmembrane helices were predicted using the TMHMM server (http://www.cbs .dtu.dk/services/TMHMM/), and signal peptides were predicted using the SignalP (version 4.1) server (http://www.cbs.dtu.dk/services /SignalP/). Tandem direct repeats were detected using the Tandem Repeats Finder program.

Protein sequence analysis and phylogenetic trees. For detection of protein sequence similarity, the nonredundant protein sequence data-base at the National Center for Biotechnology Information (NIH, Bethesda, MD) was searched using the PSI-BLAST program (37). Predicted proteins of SGPV were assigned to clusters of nucleocytoplasmic virus orthologous genes (NCVOGs) using the PSI-COG-NITOR program as previously described (38, 39). For phylogenetic analysis, protein sequences were aligned using the MUSCLE program (40) (http://www.ncbi.nlm.nih.gov/pubmed/15034147), and columns containing a large fraction of gaps (greater than 30%) and columns with low information content were removed from the alignment. The alignment was used to construct an initial maximum likelihood (ML) phylogenetic tree with the FastTree program (http://www.ncbi .nlm.nih.gov/pubmed/20224823) with default parameters. The initial tree and the alignment were fed to the ProtTest program (41) to select the best substitution matrix. For each protein family, the best matrix found by ProtTest was used to construct the final ML tree with the TreeFinder program (42).

For the construction of the phylogenetic tree of poxviruses, multiple-sequence alignments of the sequences of 13 core genes present in all Poxviridae and African swine fever virus (ASFV) were employed. These genes belong to the following NCVOGs: NCVOG0022, major capsid protein; NCVOG0023, a D5-like helicase-primase; NCVOG0031, unclassified DEAD/SNF2-like helicases; NCVOG0038, DNA polymerase elongation subunit family B; NCVOG0076, DNA or RNA helicases of superfamily II; NCVOG0249, packaging ATPase; NCVOG0261, poxvirus early transcription factor (VETF), large subunit; NCVOG0262, poxvirus late transcription factor VLTF-3-like; NCVOG0267, RNA helicase DExH-NPH-II; NCVOG0271, DNA-directed RNA polymerase subunit beta; NCVOG0274, DNA-directed RNA polymerase subunit alpha; NCVOG1117, mRNA capping enzyme; NCVOG1164, A1L late transcription factor VLTF-2.

Reconstruction of gene content evolution. The tree reconstructed from the concatenated alignment of 13 conserved proteins and the pattern of the presenceabsence of SGPV proteins in the current version of the NCVOGs (38) were used to infer the gene loss and gene gain events and to obtain an ML reconstruction of the ancestral gene sets using COUNT software (43), as previously described (39).

Genome synteny analysis. Genome synteny was visualized either with the Artemis genome comparison tool (44) or as dot plots of orthologous gene hits ordered by their positions in the genome (45). The synteny distance between viral genomes was calculated as previously described (45), with minor modifications, and a synteny-based neighbor-joining tree of the Poxviridae was constructed using the Neighbor program in the Phylip package (46).

Nucleotide sequence accession numbers. The complete sequence of the SGPV genome was deposited in GenBank under accession number KT159937.

RESULTS

Evidence for poxvirus infection in farmed salmon. The RNA isolated from salmon gill tissue containing poxvirus-like particles included sequences encoding putative proteins with significant similarity to those of poxviruses. A PCR probe was made using one such sequence in order to identify tissue with a high viral DNA content for direct paired-end sequencing. De novo assembly was performed, and gaps were filled in by PCR to generate a unique genome of 241,564 bp, excluding the termini, which were presumed to be covalently closed hairpins, as in other poxviruses. The relationship of the SGPV genome to the genomes of other poxviruses is detailed below.

TABLE 3. Overview of results.

Clinical stage	No. of fish	Median (range) CTvalue for poxvirus in gills by qPCR	IHC of gills	Hemophagocytosis
Premortality	220	18.1 (15.8-22.4)	95%	0%
Mortality	660	20.5(15.7-28.9)	91.4%*	66.7%
Postmortality	110	24.7 (18.9-30.7)	20%	20%
*2 dead fish were not sui	ted for IHC because of autolysis.

PCR and peptide antibody probes were constructed from SGPV homologs of the highly conserved vaccinia virus D13L gene and L1R virion membrane protein, respectively. Poxvirus DNA was detected by PCR in the gills from all fish sampled from the three 5 outbreak farms, with a trend of increasing Ct values over the disease course being detected (Table 3). In fish removed from tanks 1 to 3 days before death occurred (premortality stage), no lesions were found on autopsy, but most fish had no food in the gut, indicating appetite loss. On histopathology, changes were only found in the gills. Already at this stage, before clinical disease, apoptosis of lamellar epithelial cells were 10 consistently found (Fig. 1b and c). Also a general, moderate hypertrophy of this simple squamous epithelium was present, but no major blocking of the respiratory surfaces was found (Fig. 1 b). A sparse fusion of lamellae due to epithelial proliferation, and a moderate increase in the number of chloride cells were found in a few fish. All gills but one were IHC positive (Table 3). Only apoptotic epithelial cells were stained for poxvirus antigen, either 15 in the cytoplasm or in budding processes stained positive for pox-virus antigen (Fig. 3d and e). PCR of spleen and kidney tissue gave no C_T value for four fish, while one fish had a CT value of 33.8 for spleen tissue and a C_T value of 34.4 for kidney tissue. All skin tissue PCRs were positive, with the median C_T value being 29.7 (range, 23.2 to 32.6).

In tanks in which fish were lethargic and there was some mortality (mortality stage), the 20 main autopsy findings were swollen and slightly pale gills. Internal organs were also often pale, some spleens were enlarged, and no feed was found in the gut. Histopathology showed gill apoptosis in all fish at this stage, as described in the premortality stage (Fig. 3b to e). In addition, more severe gill changes obstructing the respiratory area were present in two different ways. First, in the phase with the severe detachment of apoptotic epithelial cells, the widespread adherence of the thin gill lamellae closed the water-filled space for gas exchange in an atel-ectasis-like manner (Fig. 3f). Second, the water-filled space between lamellae was solidified by proliferating epithelial cells (Fig. 3g), as demonstrated by PCNA staining (Fig. 3h). The proliferation also disrupted the tissue organization of the chloride cells (Fig. 3i), and apoptosis of chloride cells was also found. Histopathological lesions were also present in the spleen, kidney, and liver. A pronounced hemophagocytosis by scavenger endothelial cells and macrophages was found in the hematopoietic tissue of the spleen and kidney (Fig. 3l). Tissues with hemophagocytosis stained positive for Prussian blue Fe(III), demonstrating hemosiderosis (Table 3). Degenerative liver changes were variable but consistently present in dead fish. On IHC, over 90% of the gills were positive (Table 3), and labeling appeared as it did in the early stages (Fig. 3d and e). Furthermore, TEM demonstrated poxvirus-like particles in apoptotic cells (Fig. 3k). Crescents, spherical immature virions, and mature virions were seen in the cytoplasm, and these were also present in the extracellular space. Spleen and kidney were PCR negative in 3/5 fish (C_T value range, 32.6 to 35.8). All skin samples were PCR positive, with the median C_T value being 27.1 (range, 22.1 to 31.1).

In tanks in which mortality was observed a week prior to sampling (postmortality stage), most fish had no lesions on autopsy, except one fish had pale gills and four fish had enlarged spleens. Only a minor proliferation of gill epithelial cells, a very few apoptotic cells, and no IHC-positive cells were detected in all except two fish (Table 3). These two fish had pathology similar to that at the premortality stage, showing prevalent apoptosis, IHC-positive cells, and markedly lower C_T values (18.9 and 19.5) than the other fish. We also observed hemophagocytosis in the spleen and kidney at this stage, although to a much lower degree and in fewer fish than in the mortality stage (Table 3). All samples from the control cases with no signs of gill epithelial apoptosis were PCR negative, but a wide range of other gill pathologies as well as evidence for bacterial, fungal, and parasitic infections were present in the unhealthy fish (Table 4).

TABLE 4. Overview of gill lesions in the controls (no gill apoptosis)

Farm ID	Number of fish	Gill histopathology and visible agents on light microscopy
D	9	Moderate adherences of lamellae and parasitic flagellates (Ichtyobodo spp.)
E	4	Moderate detachment of lamellar epithelial cells
F	4	Moderately thickened lamellae due to epithelial hypertrophy
G	4	Severe epithelial proliferation
H	4	Moderate lifting of epithelial cells and fungal infection
I	3	Moderate epithelial hypertrophy
J	4	Severe epithelial proliferation
K	10	Focal detachment and necrosis of lamellar epithelial cells and bacteria colonizing the apical surface of epithelium
L	6	Moderate hypertrophy and necrosis of epithelial cells; mucous cell proliferation.

From each of the 14 archived, formalin-fixed, paraffin-embedded case series, at least one positive gill tissue sample was found by PCR. The 39 diseased fish had a median CT value of 25.9 (range, 20.1 to 36.2). The interspersed control tissues had either high C_T values or no C_T value, indicating low or no cross contamination. All 12 samples from the so-called amoebic gill disease case were positive for poxvirus DNA by qPCR.

Genome analysis and evolutionary relationships of SGPV. The SGPV genome consists of 241,564 bp (excluding the terminal hairpins) with a 37.5% GC content. The genome contains inverted terminal repeats of 5,679 bp each, similar to other poxviruses.

Each of the inverted repeats, in turn, encompasses arrays of direct repeats. However, the tandem direct repeat arrays of SGPV, located at the very ends of the available genomic sequence, consist of only two 89-bp repeat units with 90% identity matches (each of these units consists of two 45-bp repeats with 88% identity). Thus, these direct repeat arrays are much smaller than those detected in other chordopoxviruses, although the possibility that they extend beyond the sequenced portion of the genome cannot be ruled out. Indeed, the highly conserved concatemer resolution sequence that is located between the repeat array and the apex of the terminal hairpin in other poxvirus genomes was not detected at the ends of the available SGPV sequence. The SGPV genome encompasses 206 unique predicted protein-coding genes (4 of these are contained within the terminal repeats and, accordingly, are present in the genome in two copies each; for details on gene prediction, see Materials and Methods). Comparison of the protein sequences encoded by these predicted genes to the sequences in the nonredundant protein sequence database at the National Center for Biotechnology Information (NIH, Bethesda, MD) using PSI-BLAST identified homologs with significant sequence similarity (E values, <104) for only 60 genes (for several additional predicted proteins, hits with apparently significant E values were identified as originating from regions of low sequence complexity and, accordingly, were dismissed as spurious). In addition to the standard database search, the predicted SGPV protein sequences were compared to the sequences of the NCVOGs (39), clusters of orthologous genes of nucleocytoplasmic large DNA viruses (NCLDVs), using a sequence profile search (see Materials and Methods). This comparison resulted in the assignment of 68 SGPV genes to NCVOGs, including 6 genes that showed no significant similarity to other proteins in BLAST searches. Additionally, a search for conserved domains led to a functional prediction in yet another protein (SGPV102). In total, specific, sequence conservation-based annotations were obtained through these procedures for 71 (34%) SGPV genes (Table 5 and Fig. 3). Among the genes without detectable homologs, 23 contained predicted trans-membrane segments and/or a signal peptide, whereas 111 genes (55%) remained completely uncharacterized. Among the predicted products of these uncharacterized genes, many primarily consisted of low-complexity sequences and/or contained simple amino acid repeats (Table 5). These proteins are likely to be structurally disordered or assume unusual tertiary structures.

Among the predicted gene products of SGPV, homologs in other chordopoxviruses were detected for 59 proteins (Table 5 and Fig. 4). Among these conserved chordopoxvirus proteins, 32 belong to the previously inferred ancestral NCLDV gene set (38, 47), 17 are represented in all poxviruses (including entomopoxviruses), and 10 are specific for chordopoxviruses (Table 5 and Fig. 4). Eight genes have homologs in other NCLDVs but most likely were acquired independently (convergently), as suggested by sequence similarity and phylogenetic analysis, and only 4 genes appear to represent unique genes (with respect to the NCLDVs) captured from cellular organisms (Table 5 and Fig. 4; see Discussion below).

The conserved gene set includes most of the essential genes involved in virus DNA replication and expression as well as the morphogenesis and structure of the virion core and the capsid (see the discussion of some notable exceptions in “Shared and distinct gene functions between SGPV and other chordopoxviruses and unexpected evolutionary patterns among SGPV genes” below). All conserved genes of SGPV showed the highest sequence similarity to the orthologs from chordopoxviruses, with only 3 exceptions, where the highest similarity (albeit by a small margin) was observed with entomopoxvirus orthologs (Table 5). These observations imply that the conserved SGPV genes share an evolutionary history, at least within the poxviruses. Accordingly, we used concatenated multiple-sequence alignments of the sequences of 13 highly conserved genes from this ancestral gene set to construct a maximum likelihood (ML) phylogenetic tree in which the root was placed between ASFV and the poxviruses, given that ASFV and poxviruses are sister groups in the overall NCLDV phylogeny (39). In the resulting tree, SGPV was placed at the root of the chordopoxvirus branch with unequivocal bootstrap support (Fig. 5). Thus, the phylogeny of chordopoxviruses generally follows the phylogeny of their hosts.

In order to gain further insight into the evolution of the gene complement of SGPV, we performed an ML reconstruction of the ancestral gene sets using the poxvirus phylogenetic tree (Fig. 5) as a guide. The inferred ancestral gene sets showed an unexpected pattern (Fig. 6): 58 genes were mapped to the common ancestor of all poxviruses, and 62 genes were mapped to the common ancestor of chordopoxviruses. Thus, taken in their entirety, chordopoxviruses possess almost the same conserved gene set as the entire family Poxviridae, with very few additional conserved genes appearing after the divergence from the common ancestor with entomopoxviruses. In contrast, 38 additional genes were mapped to the common ancestor of the chordopoxviruses infecting tetrapods; i.e., these genes were gained along the tree branch between SGPV and crocodile poxvirus (CrPV). Thus, the reconstruction reveals a dramatic difference in the conserved gene repertoires between the common ancestor of all chordopoxviruses and the tetrapod poxvirus ancestor (Fig. 6). This difference likely reflects a major biological transition, the possible nature of which is discussed in “Shared and distinct gene functions between SGPV and other chordopoxviruses and unexpected evolutionary patterns among SGPV genes” below.

SGPV gene 001 002 003

004 005 006 007 008

009

010

011

012 013 014 015 016 017 018 019 020 021 022 023 024 025 026 027 028 029 030 031

032

033

034

035

036

037

038

039

040

041

042

043

044

045

Genome coordinates (protein length⁰)

VACV gene name

Best hit (GI|Eval|% identity|aln len|organism)

NCVOG Representation among no NCLDVs

Predicted TM Functional annotation, comments, and SP or inferred origin

TABLE 5 Predicted genes of SGPV^a

1248-298 (317)	Hypothetical protein
2205-1288 (306)					Hypothetical protein
3227-2241 (329)					Hypothetical protein; low
					sequence complexity
4788-3361 (476)					Hypothetical protein
5884-4934 (317)					Hypothetical protein
7314-6361 (318)					Hypothetical protein
7770-7438 (111)					Hypothetical protein
8702-7830 (291)					Hypothetical protein
9383-9054 (110)				1 TM (C)	Hypothetical type I membrane
					protein, heptad repeats
9911-9546 (122)					Hypothetical protein; low
					sequence complexity
11295-10021 (425)					Hypothetical protein; low
					sequence complexity
12365-11373 (331)					Hypothetical protein
13629-12421 (403)					Hypothetical protein
15099-13681 (473)					Hypothetical protein
15912-15166 (249)					Hypothetical protein
16318-16007 (104)					Hypothetical protein
17728-16409 (440)					Hypothetical protein
18401-17922 (160)					Hypothetical protein
18870-18373 (166)					Hypothetical protein
19147-18863 (95)					Hypothetical protein
19468-19196 (91)					Hypothetical protein
19721-19533 (63)				1 TM (M)	Hypothetical membrane protein
20272-19751 (174)					Hypothetical protein
21336-20332 (335)					Hypothetical protein
21953-21345 (203)					Hypothetical protein
23067-21967 (367)					Hypothetical protein
24336-23167 (390)					Hypothetical protein
25255-24395 (287)					Hypothetical protein
25986-25294 (231)					Hypothetical protein
26353-26033 (107)					Hypothetical protein
26850-26389 (154)				1 TM (C)	Hypothetical type I membrane
					protein
27837-26851 (329)	0017	Phy, Mimi, Ent	401825817|3 E— 11\28\180\Encephalitozoon		Λ'-Myristoyl transferase; probable
			hellem ATCC 50504		independent acquisition in different viruses; CACQ
28446-27847 (200)					Hypothetical protein
28512-30752 (747)	0330	Most NCLDVs, all	660515722\3E-07\26\242\Armadillidium		Divergent RING finger protein,
		families except Asf	vulgare iridescent virus		potential E3 subunit of ubiquitin ligase; uncharacterized N-terminal domain upstream of RING domain; RING proteins in different NCLDVs likely have different origins; this SGPV protein is most similar to homologs from Iri and Phy;
					CACQ
31420-30749 (224)					Hypothetical protein
32306-31434 (291)					Hypothetical protein
34172-32325 (616)					Hypothetical protein
34466-34263 (68)				SP	Hypothetical protein
34829-34503 (109)					Hypothetical protein
35364-34792 (191)	0202	Pox, Iri	617520525|5 E-06|30|123|Poecilia formosa	1 TM (C), SP	Ig domain type I membrane
					protein; not closely related to Ig domain-containing proteins of other NCLDVs; CACQ
35957-35583 (125)					Hypothetical protein
36110-36592 (161)				1 TM	Hypothetical protein
36828-37679 (284)	0284	Some representatives	511086842|1 E-59|39|276|EHtamoeba		Ser/Thr protein kinase; probable
		of most NCLDVs	histolytica		eukaryotic origin; putative
		except Asf			ribosomal protein S6K, mTOR pathway component; not closely related to any other NCLDV kinase, likely independent origin; CACQ
38096-37665 (144)				1 TM (M)	Hypothetical membrane protein
39115-38135 (327)	1068	Scattered distribution F2L	254568556|8E-14|33|141|Komagataella	1 TM (N)	Trimeric dUTPase highly similar
		in all NCLDV	pastoris GS115		to homologs from Phy but not
		families			poxviruses; contains uncharacterized N-terminal
					domain with a predicted TM; ANC

(Continued on following page)

	VACV
Genome coordinates (protein length0)	NCVOG Representation among gene Best hit (GI|Eval|% Predicted TM Functional annotation, comments, no NCLDVs name identity|aln len|organism) and SP or inferred origin

39674-39102 (191

40162-39689 (158)

40352-40882 (177)

41988-40891 (366)

42947-42033 (305)

45667-42950 (906)

46422-45664 (253)

46580-46422 (53)

47190-46636 (185)

48431-47325 (369)

49116-48292 (275) 50255-49101 (385) 52022-50265 (586) 52040-52516 (159)

1122 All Pox, some Iri, Mimi J5

53257-52439 (273) 53859-53299 (187)	0258	All Chor	J4R
54821-53886 (312)	1152	All Pox, Pith, some	J3R
		Mimi
55426-54788 (213)
55824-55390 (145)
56600-55842 (253)	1063	All Pox	L4R
56631-57599 (323)	1168	All Pox	L3L
59023-57596 (476)	0295	Most NCLDV families	F10L

except Asf and Pan

59043-59897 (285)	0249	All NCLDVs	A32L
59901-60317 (139) 62029-60323 (569)	1165	All Pox	E1L
62691-62071 (207)	0272	All Pox, most other	E4L
		NCLDVs
63448-62681 (256) 64802-63516 (429) 64862-66538 (559)	1173	All Pox	E6R
66539-67645 (369)
70831-67634 (1,066)	0038	All NCLDVs	E9L
71147-70848 (100)	0052	All NLCDVs	E10R

71159-71788 (210) 72152-71760 (131) 73310-72168 (381)	1160	All Chor	I1L
74930-73677 (418)
76048-74933 (372)	1171	All Chor	I6L

77179-76049 (377)

77295-78926 (544)

79246-78857 (130)

81234-79657 (526)

81913-81221 (231)

81912-82319 (136)

089

86171-82275 (1,299)

0190

502875360|8E-4|24|403|Rlanctomyces limnophilus

TM (C)

TM (C)

TM (N)

TM

13876678|1E-08|31|186|lumpy skin disease virus

41057489|3E-42|36|276|bovine papular stomatitis virus

TM (M)

659488262|5 E-08|24|308|penguinpox virus

544837|6 E-26|28|396|variola virus VAR,

India-1967, peptide, 439 amino acids

12085104|1 E-17|28|264|Yaba-like disease virus

9631476\2E-14\23\381\Melanoplus sanguinipes entomopoxvirus 38229198|5 E-18|29|174|Yaba monkey tumor virus

40556061|3E-07|19|547|canarypox virus

659488229|5E-142|32|1027|penguinpox virus

40556058|3E-24|48|92|canarypox virus 1 TM (false positive)

5830616|4E-13|27|281|variola minor virus

TM

Hypothetical protein

Hypothetical protein

Hypothetical protein

Hypothetical protein

Hypothetical protein

Metalloendopeptidase of the M60like family; UAQ

Hypothetical type I membrane protein

Hypothetical type I membrane protein

Hypothetical type II membrane protein

Hypothetical protein

Hypothetical protein

Hypothetical protein

Hypothetical protein

Myristylated membrane protein, entry-fusion complex subunit;

ANC; TAAATG

Hypothetical protein

DNA-dependent RNA polymerase subunit Rpo22; CPOX

Poly(A) polymerase small subunit, cap O-methyltransferase; ANC

Hypothetical protein

Hypothetical membrane protein DNA-binding virion core protein

VP8; POX; TAAATG

Virion protein required for early transcription; POX

Protein kinase involved in early stages of virion morphogenesis;

ANC

DNA packaging ATPase; ANC; TAAATG

Hypothetical protein

Poxvirus poly(A) polymerase catalytic subunit POX

Transcription factor S-II (TFIIS); ANC; TAAATG

Hypothetical protein

Hypothetical protein

Virion protein required for the formation of mature virions;

POX; TAAATG

Hypothetical protein; low sequence complexity

DNA polymerase ANC

Disulfide (thiol) oxidoreductase (Erv1/Alr family) involved in disulfide bond formation during virion morphogenesis;

ANC; TAAATG

Hypothetical protein

Hypothetical protein

DNA-binding virion core protein;

CPOX

Hypothetical protein Telomere-binding protein involved in viral DNA encapsidation; CPOX;

TAAATG

Hypothetical protein

Hypothetical protein

Hypothetical protein; low sequence complexity

Hypothetical protein; low sequence complexity

Hypothetical protein; low sequence complexity

Protein consists of hydrophobic decamer repeats; TM prediction could be spurious

Hypothetical protein (Continued on following page)

Genome coordinates (protein length0)	NCVOG no	Representation among NCLDVs	VACV gene name	Best hit (GI|Eval|% identity|aln len|organism)	Predicted TM and SP	Functional annotation, comments, or inferred origin
88408-86198 (737)	0031	Nearly all NCLDVs	D6R	345107280|1E---156|41|657|Yoka poxvirus		SNF2-like helicase involved in
						early transcription; ANC
90927-88453 (825)	0023	All NCLDVs	D5R	571798002|5E---93|28|768|squirrelpox		Primase-helicase; ANC
				virus
91684-90920 (255)	0211	All Chor	F9L	9634782|8E-06|33|123|fowlpox virus	1 TM (C)	Myristylated IMV envelope
						protein; CPOX; TAAATG
92207-91647 (187)						Hypothetical protein
92262-92876 (205)	1067	Mimi		494264790|3E---09|28| 11‘8\/Marinobacter		Deoxynucleotide monophosphate
				algicola		kinase shared with Mimi, probable bacterial origin; CACQ
96496-93050 (1,149)	0037	Phy, Mimi, Mar, CrPV		5121|2E—53|25|933| Schizosaccharomyces		DNA topoisomerase II; ANC
		(multiple paralogs) but not other Pox		pombe
96825-96496 (110)					1 TM (M)	Hypothetical membrane protein
97628-96828 (267)	0211	Most NCLDVs, all Pox	L1R	12085043|2E---29|31|225|Yaba-like	1 TM (C)	Myristylated IMV envelope
				disease virus		protein; ANC; TAAATG
99427-97661 (589)	0022	All NCLDVs except Pan	D13L	345107288|8E-50|28|570|Yoka poxvirus		Major capsid protein (involved in
						morphogenesis but not incorporated into virions in poxviruses); ANC
99845-99456 (130)	1164	All NCLDV	A1L	289183841|1E-12|29|123|pseudocowpox		Late transcription factor VLTF-2;
				virus		ANC
100665-99853 (271)	0262	All NCLDVs except	A2L	571798015|7E-8|38|195|squirrelpox virus		Late transcription factor VLTF;
		Pith				ANC
103192-100967 (742)	1162	All Pox, Mimi	A3L	115531788|1E-49|24|697|Nile		Poxvirus P4B major core protein;
				crocodilepox virus		POX
103950-103237 (238)		MCV, some Mimi, Phy				J domain-containing protein,
						putative cochaperonin; distantly related to J domains of other
						NCLDVs; CACQ
103956-104498 (181)	1377	All Chor	A5R	40556180|3E-14|33|172|canarypox virus		DNA-dependent RNA polymerase
						subunit Rpo19; CPOX
105738-104503 (412)	1179	All Chor	A6L			Virion core protein required for
						membrane biogenesis and formation of mature virions;
						CPOX; TAAATG
107964-105751 (738)	0261	All Pox, scattered in	A7L	659488305|2E-104|32|734|penguinpox		VETF, large subunit; ANC
		other NCLDVs		virus
107945-109252 (436)	1176	All Chor	A8R	40556183|2E-08|24|248|canarypox virus		Poxvirus intermediate
						transcription factor VITF-3 subunit; CPOX; TAAATG
109518-109261 (86)					2 TM	Hypothetical membrane protein
113053-109535 (1,173)	0257	All Pox	A10L	157939724|4E-13|20|561|tanapox virus		Virion core protein P4; POX;
						TAAATG
113084-113923 (280)						Hypothetical protein
113964-114296 (111)					1 TM (N)	Hypothetical type II membrane
						protein
114326-114619 (98)						Hypothetical protein; low
						sequence complexity
114935-114600 (112)						Hypothetical protein containing
						serine-rich repeats
115188-115556 (123)						Hypothetical protein
115768-115556 (71)					1 TM (N)	Hypothetical type II membrane
						protein
117013-115769 (415)	1045	Some Iri and Mimi		339906034|2E---07|30|145|Wiseana		5 -3 exoribonuclease of the XRN
				iridescent virus		family; NCLDV proteins appear to be monophyletic; ANC
117383-117045 (113)					2 TM	Hypothetical protein
117721-117401 (107)						Hypothetical protein
118830-117736 (365)	1122	All Pox, Mimi, some Iri	A16L	41057529|2E-15|29|204|bovine	1 TM	Myristylated protein, entry-fusion
				papular stomatitis virus		complex subunit; ANC; TAAATG
119936-118848 (363)					2 TM	Hypothetical membrane protein
119988-121418 (477)	0076	All Pox, in many other	A18R	115531805|1E-54|29|424|Nile		DNA helicase of superfamily 2,
		NCLDVs		crocodilepox virus		transcript release factor; ANC
121419-122474 (352)	2643	Some Mimi		504603808|3E-15|30|151|		Apurinic-apyrimidinic
				Ornithobacterium rhinotracheale		endonuclease of the exonuclease III family; probable bacterial origin; CACQ
122812-122465 (116)	1370	All Pox	A21L	506498863|2E---06\24\111\Choris!oneura	1 TM (C)	Type I membrane protein, entry-

rosaceana entomopoxvirus L fusion complex subunit; POX;

TAAATG (Continued on following page)

VACV Genome coordinates NCVOG Representation among gene (protein length⁰) no NCLDVs name

122842-125133 (764) 0035 CrPV, Ent, some Iri,

Mimi

125105-125602 (166)	0278	All Pox, majority of	A22R
		other NCLDVs
125599-126816 (406)	0263	All Pox	A23R
126817-130305 (1,163)	0271	All NCLDVs except	A24R
		some Phy
130720-130310 (137)	1418	All Pox	A28L

131699-130725 (325)	0260	All Pox	A29L
131870-132817 (316) 132821-133525 (235) 133536-135035 (500) 135013-135495 (161) 135919-135470 (150)
136606-135941 (222) 136671-138380 (570)	1115	All Pox, scattered in other NCLDVs	D4R
138373-139212 (280)	0259	All Pox	D7R
139235-139879 (215)	0236	All Pox, most other NCLDVs	D10R
141785-139887 (633)	0027	All Pox, some Mimi	D11L
141949-142902 (318)	0330	All NCLDVs except Asco and Pith

143889-142951 (313)	1169	All Pox	D12L
144893-143889 (335)	1122	All Pox, some Mimi, Iri	G9R
145769-144894 (292)	1369	All Chor	G8R
145819-147291 (491)
147884-147288 (199)	1182	All Pox	G6R

148111-147914 (66)	1368	All Chor, one Ent, Asf	G5 5R
149892-148072 (607)	1060	All Pox, scattered in other NCLDVs	G5R
149931-150485 (185)
150507-150884 (126) 150881-152773 (631)	1170	All Pox	G1L
154796-152760 (679)	0267	All Pox, Asf, Mimi	I8R
154823-156076 (418)	1161	All Pox, most other NCLDVs	I7L

156073-156567 (165)

153

156623-157354(244)

Best hit (GI|Eval|% identity|aln len|organism)

659488557|6E—16|32|149|pigeonpox virus

9634858|3E—25|27|395|fowlpox virus

225194776|0|47|1169|skunkpox virus

51317191|3E— 17\33\128\Diachasmimorpha longicaudata entomopoxvirus

148912996|9E—08|27 |181|goatpox virus Pellor

9634732|1E—15|28|216|fowlpox virus

9629029|4E—17|30|145|molluscum contagiosum virus subtype 1 9629031|1E—15|29 |161|molluscum contagiosum virus subtype 1 115531782|2E—174|43|635|Nile crocodilepox virus

658035022|2E—06\31\75\Malus domestica

9629033|3E—31|30|289|molluscum contagiosum virus subtype 1

9634797|7E—06|36|78|fowlpox virus

41057481|1E—06|26|171|bovine papular stomatitis virus

289183806|2E—04|24|65|pseudocowpox virus

539191060|6E—13|36|176|myxoma virus

505137967|1E—05|41|59|

Methanomethylovorans hollandica

115531736|6E—35|31|233|Nile crocodilepox virus

41057099|5E—121|37|597|orf virus

115531734|7E—15|21|429|

Predicted TM Functional annotation, comments, and SP or inferred origin

NAD-dependent DNA ligase; poorly conserved sequence but contains intact catalytic residues and shows the closest sequence similarity to NAD-dependent ligases of Ent; ANC

RuvC family Holliday junction resolvase; ANC

Intermediate transcription factor;

POX

DNA-directed RNA polymerase subunit beta; TAAATG

Type I membrane protein, entryfusion complex subunit beta; ANC; TAAATG

DNA-directed RNA polymerase, 35-kDa subunit; POX

Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein UDG; ANC

Hypothetical protein; low sequence complexity DNA-directed RNA polymerase, 18-kDa subunit; POX

Nudix hydrolase, decapping enzyme; ANC

Superfamily 2 helicase D11; POX; TAAATG

RING finger-containing E3 ubiquitin ligase; probably independent acquisition in different NCLDV families;

CACQ

Poxvirus mRNA capping enzyme, small subunit; POX; TAAATG Myristylated protein, entry-fusion complex subunit; ANC;

TAAATG

Protein containing a derived PCNA domain; VLTF-1; CPOX;

TAAATG

Hypothetical protein Predicted hydrolase or acyltransferase of the NlpC/P60 superfamily; weak sequence similarity to orthologs in other poxviruses; POX; TAAATG RNA polymerase, subunit 10 (a very small protein, possibly missed during genome annotation of other viruses);

POX

Flap endonuclease required for poxvirus genome replication;

ANC

Thioredoxin; no close homologs in other viruses; UAQ

Hypothetical membrane protein Metalloprotease essential for virion morphogenesis; POX; TAAATG

RNA helicase of superfamily 2 implicated in early transcription termination; ANC; TAAATG Virion core cysteine protease involved in virion protein maturation; ANC; TAAATG Hypothetical protein

TM (N)

TM

TM (M)

TM (C), SP

Hypothetical protein (Continued on following page)

VACV

SGPV gene	Genome coordinates (protein length0)	NCVOG no	Representation among NCLDVs	gene name	Best hit (GI Eval % identity aln len organism)	Predicted TM and SP	Functional annotation, comments, or inferred origin
154	157464-164144 (2,227)	0269	All Chor; disrupted in	(B22R	422933904 3E-120 29 1049 cyprinid	1 TM (C), SP	Giant type I membrane protein
			some, including	VARV)	herpesvirus 2		with homologs also in cyprinid
			VACVs				herpesviruses, suggestive of gene transfer from SGPV to the herpesviruses (see the phylogenetic tree in Fig 10); implicated in T cell inactivation; paralog of SGPV159 and SGPV162; CPOX
155	164257-168030 (1,258)					1 TM (C), SP	Hypothetical type I membrane
							protein
156	168031-169008(326)						Hypothetical protein
157	168995-169900(302)						Hypothetical protein
158	170583-169939 (215)						Hypothetical protein
159	170638-173652 (1,005)	0269	All Chor; disrupted in	0	9634792 5E-11 |24|462|fowlpox virus	1 TM (C), SP	Giant type I membrane protein
			some, including				with homologs also in cyprinid
			VACV				herpesviruses, suggestive of gene transfer from SGPV to the herpesviruses (see the phylogenetic tree in Fig 10); implicated in T cell inactivation; paralog of
							SGPV154 and SGPV162; CPOX
160	173910-173665 (82)					1 TM(N)	Hypothetical type II membrane
							protein containing pentapeptide repeats
161	173870-181351 (2,494)					SP	Hypothetical secreted protein
162	181528-185433 (1,302)	0269	All Chor; disrupted in	0	9628967 5E-25 25 413 molluscum	1 TM (C), SP	Giant type I membrane protein
			some, including		contagiosum virus subtype 1		with homologs also in cyprinid
			VACVs				herpesviruses, suggestive of gene transfer from SGPV to the herpesviruses (see the phylogenetic tree in Fig 10); implicated in T cell inactivation; paralog of
							SGPV154 and SGPV159; CPOX
163	185473-186558(362)						Hypothetical protein
164	186693-188648(652)					SP	Hypothetical secreted protein
165	188749-192687 (1,313)	0274	All NCLDVs except for	J6R	1155317631014111311 |Nile crocodilepox		DNA-directed RNA polymerase
			some Phy		virus		subunit alpha; ANC
166	193271-192684(196)					1 TM(C)	Hypothetical type I membrane
							protein
167	193287-194597 (437)					SP	Hypothetical secreted protein,
						pentapeptide repeats
168	195155-194586 (190)	0253	All Pox	H2R	5 940195 9512E-3 514011511 avipoxvirus OKr-	1 TM(N)	Type II membrane protein, fusion-
					2014		entry complex subunit; POX; TAAATG
169	197626-195161 (822)	1163	All Pox	H4L	6969751 3E-67 30 583 vaccinia virus Tian		Pox Rap94, RNA polymerase-
					Tan		associated transcription specificity factor, Rap94; POX;
							TAAATG
170	197724-198404 (227)						Hypothetical protein
171	198405-199343 (313)	0036	All Pox, Mimi	H6R	345107272 5E-60 40 310 Yoka poxvirus		DNA topoisomerase IB; ANC;
							TAAATG
172	199715-199329(129)					SP	Hypothetical secreted protein
173	199747-202368 (874)	1451	All NCDLVs except	DIR	225194732 4E-110 33 867 volepox virus		mRNA capping enzyme large
			Asco and Pan				subunit; ANC; TAAATG
174	204943-202382 (854)						Hypothetical protein
175	205237-204956 (94)						Hypothetical protein
176	205654-205238 (139)						Hypothetical protein
177	205659-207647 (663)						Hypothetical protein
178	207690-209033 (448)						Hypothetical protein
179	209178-209951(258)						Hypothetical protein
180	210027-211280(418)						Hypothetical protein
181	211532-213193 (554)						Hypothetical protein
182	213211-213954 (248)						Hypothetical protein
183	213947-214258 (104)						Hypothetical protein
184	214236-214847(204)						Hypothetical protein
185	215300-214851 (150)						Hypothetical protein
186	215396-216664 (423)				167525479 6E-18 28 228|A/oHO5igti		DNA or RNA methyltransferase;
					brevicollis MX1		UAQ
187	216775-217242 (156)				20973420819E-291461127|^/wo salar		Macrodomain, most similar to
							<9-acetyl-ADP-ribose deacetylase; UAQ
188	217294-218286 (331)						Hypothetical protein
189	218360-219514(385)						Hypothetical protein

(Continued on following page)

VACV

SGPV Genome coordinates NCVOG Representation among gene Best hit (GI|Eval|% Predicted TM Functional annotation, comments, gene (protein length⁰) no NCLDVs name identity|aln len|organism) and SP or inferred origin

190	219572-220492 (307)	Hypothetical protein; low
191	220576-221535 (320)	sequence complexity; partly consists of tetrapeptide repeats Hypothetical protein
192	221579-222580 (334)	Hypothetical protein
193	222676-223716 (347)	Hypothetical protein
194	224007-224258 (84)	Hypothetical protein;
195	224390-225718 (443)	hydrophobic; 12-mer repeats Hypothetical protein; low
196	226126-226542 (139)	sequence complexity Hypothetical protein
197	226596-227135 (180)	Hypothetical protein; cysteine
198	227202-228812 (537)	rich; low sequence complexity Hypothetical protein; low
199	228872-229897 (342)	sequence complexity Hypothetical protein
200	229951-230268 (106)	Hypothetical protein
201	230293-230985 (231)	Hypothetical protein
202	231098-231802 (235)	Hypothetical protein
203	232049-233533 (495)	Hypothetical protein
204	233892-233491 (134)	4 TM Hypothetical protein;
205	234526-235545 (340)	hydrophobic, consists mostly of hexapeptide repeats; TM prediction might be false positive Hypothetical protein
206	235633-236631 (333)	Hypothetical protein
207	236777-238204 (476)	Inverted terminal repeat; identical
208	238338-239324 (329)	to SGPV001 gene Inverted terminal repeat; identical
209	239360-240277 (306)	to SGPV002 gene Inverted terminal repeat; identical
210	240317-241267 (317)	to SGPV003 gene Inverted terminal repeat; identical

to SGPV004 gene ^a In the first column, “SGPV” is omitted from the gene identifiers for brevity; in the last column “SGPV” is included; GI, GenInfo Identifier sequence identification number); aln_len, the length of pairwise protein alignment produced by BLASTP searches; SP, (predicted) signal peptide; TM, (predicted) transmembrane 5 helix (C, M, and N denote the C-terminal, middle, and N-terminal location of the predicted transmembrane helix in the protein, respectively); the percent identity and alignment length are taken directly from BLASTP searches. IMV stands for intracellular mature virions; VARV stands for variola virus. The inferred origin of genes is indicated as follows: ANC, ancestral to NCLDV; POX, ancestral to poxviruses; CPOX, ancestral to chordopoxviruses; CACQ, convergent acquisition (with other NCLDVs); UAQ, unique acquisition. The transcription start element TAAAT is shown for those SGPV genes that have orthologs from other chordopoxviruses (the sequence TAAATG includes the translation start codon of the respective gene). Abbreviations for groups of viruses: Asco, Ascoviridae; Asf, Asfarviridae; Chor; Chordopoxvirinae; CrPV, crocodile poxvirus; Ent, Entomopoxvirinae; Iri, Iridoviridae; Mar, Marseilleviridae; MCV, molluscum contagiosum virus; Mimi, Mimiviridae; Pan, Pandoravirus; Phy, Phycodnaviridae; Pith, Pithovirus; Pox, poxviruses.

^b Protein lengths are in numbers of amino acids.

The tetrapod chordopoxviruses, except for avipoxviruses, are characterized by a distinct genome architecture whereby the central portion of the genome shows a nearly perfect conservation of gene synteny and the terminal regions are highly divergent and often 20 contain unique genes, as depicted in the dot plots of Fig. 7. In contrast, genome-wide comparison of the gene orders between SGPV and other chordopoxviruses shows the extensive decay of synteny in SGPV and the complete disappearance of synteny between chordopoxviruses and entomopoxviruses (Fig. 7). Examination of the genomic dot plots (Fig. 7) and a genome architecture alignment (Fig. 8) between SGPV and other chordopoxviruses reveals several conserved gene blocks in the central part of the genome that are separated by strings of nonhomologous genes of variable length, along with at least two inversions of conserved genomic segments. To assess the evolution of the pox-virus genome architecture in more quantitative terms, we calculated the matrix of genome rearrangement distances and used it to construct an evolutionary tree of genome architectures (Fig. 9). This tree shows that the decay of synteny roughly follows the evolution of gene sequences (compare the trees in Fig. 9 and 5), but the rate of disruption of the ancestral gene order is nonuniform, with the major change mapping to the branch between SGPV and the rest of the chordopoxviruses.

Shared and distinct gene functions between SGPV and other chordopoxviruses and unexpected evolutionary patterns among SGPV genes. Here we discuss the predicted functions and some unusual aspects of evolution of the SGPV genes in the order of the tiers of ancestry, i.e., the point of gene origin for (acquisition by) this virus (Fig. 4). The ancestral NCLDV genes retained by SGPV encode the principal functions required for genome replication and expression, with no genes having been lost since the common ancestor of all poxviruses. However, two genes merit special mention in the context of poxvirus evolution, namely, the genes for DNA topoisomerase II (Topo II; SGPV095) and NAD-dependent DNA ligase (SGPV123). These (putative) ancestral NCLDV genes are uncharacteristic of chordopoxviruses, being present only in CrPV and SGPV, whose genomes encode both Topo II (which has multiple paralogs in CrPV) and Topo IB, which is conserved in the rest of the chordopoxviruses. The evolution of topoisomerases in NCLDVs appears to have been quite complex, involving both differential gene loss and the apparent independent acquisition of homologous genes. Thus, SGPV and CrPV could represent either the ancestral state with two distinct topoisomerase genes or an intermediate state after the Topo IB gene had been acquired but the Topo II gene had not been lost as it was in chordopoxviruses and entomopoxviruses independently.

The NAD-dependent ligase also appears to be an ancestral NCLDV gene but was replaced by the distinct, ATP-dependent ligase in several groups of viruses, including most of the chordo-poxviruses, after the divergence from the common ancestor with CrPV. The finding that SGPV encodes an NAD-dependent ligase but not an ATPDK 180123 B1 dependent ligase is compatible with this scenario. The predicted NAD-dependent ligase of SGPV shows an unexpectedly low sequence similarity to homologs from other NCLDVs (Table 5), suggestive of some peculiarity in the DNA replication process of this virus.

The next evolutionary tier of the SGPV genes, those that are conserved in all poxviruses (Table 6), includes components of the transcription apparatus, such as several RNA polymerase subunits and the poly(A) polymerase catalytic subunit; several components of the virion core and proteins involved in virion morphogenesis, such as the metalloprotease G1; and six subunits of the fusion-entry complex (homologs of three paralogous subunits of this complex, A16, G9, and J5, are also detectable in mimiviruses and iridoviruses, suggesting that some form of this complex might be ancestral in NCLDVs). Of note is the presence in SGPV of a highly diverged ortholog of the G6 protein, a predicted amidase or acyltransferase that is thought to be important for the virus-host interaction but whose specific function remains obscure.

The genes that are conserved in chordopoxviruses, to the exclusion of entomopoxviruses, follow the same general functional themes, including RNA polymerase subunits, such as A5 and J4; the late-stage transcription factor G8 containing a highly diverged PCNA domain; and proteins involved in core morphogenesis, e.g., the telomere-binding protein I6 and the protein A6 required for virus membrane biogenesis. Particularly notable in this group are three paralogous genes (SGPV154, SGPV159, SGPV162), located near the right end of the genome, that encode homologs of variola virus B22R, a giant type I membrane protein implicated in the virus-induced shutdown of the host adaptive immunity, specifically, inhibition of T lymphocytes. Of these three paralogous genes, SGPV154 is similar in length to homologs from other poxviruses, whereas SGPV159 and SGPV162 are considerably shorter and, thus, have apparently been truncated during the evolution of the SGPV lineage. However, the conservation of the predicted signal peptide and the C-terminal transmembrane helix in all three proteins (Table 5) suggests that they remain functional. The proliferation of this gene in SGPV, which parallels its independent triplication in CrPV, implies an important role of this route of counterdefense. Interestingly, homologs of this gene were also detected in cyprinid herpesviruses, suggesting transfer of this gene, involved in virus-host interaction from SGPV (or its relative), to unrelated viruses within the same host (Fig. 10). Moreover, the cluster of SGPV genes that encompasses the three B22R paralogs also contains two other proteins with a similar, very large size (SGPV155 and SGPV161; Table 5) that are predicted to be, respectively, membrane associated and secreted. The sequences of these proteins show no similarity to the B22R sequence, but the proteins might perform roles similar to the role performed by B22R via a distinct mechanism.

TABLE 6 Conserved chordopoxvirus genes missing in SGPV:

Conserved gene*	VaCV jene	Kno^n or predkred fantrion	Essential*	Comment
Genet «·η«ηο1 inehordopoxvinn« ami earrænopovinise*
II7S	Läfi	Membrane protein, farion-enny campten subunit	Ϊ·«
1 IHI	AllR	Membrane-ass«ia«d protein imp! kated in endoplasmk rerkidum recruitment (or virion nwrpbojenesis	f-w
	HlL	Dnal-spocifeiry ·:3ετΑΠιτ and Tyr'l protein phosphatase	f-w	Coftwrvcxl in onfriv*o wiTonwpoKiiniM*
Gene* «mtervol only in thordopoitvinrws.
IIH5	ajüR	D:J A polymerase prwewivity factor	Ϊ-Η
IMS	I'L	Single-stranded (WA-binding protein essential for repikstion
IIH4	G1R	Late transer i pt ion dongat k*i ireox
1171	AI.L	Virion «weprwein	f-W
1177	F1?R	DlJA-bindingvirwn core protein
KUJ	G7l	Virion «weprwein required for immature virion form*™	f-W
Ι3Ή	AHL	Virion core protein
	G4L	Glinaredonin involved in the pattitvay forcytoplasmk diuMlklf bond (onrut kn	f-W
iMt.	A2-5L	Thieredoxin-lilre protein involved indw pathway for eyioplasm k disid fido bond fornurion	f-W
WL2	AMR	C-npe lectin involved in ™i»«War virion morphogenesis	>!O
Q2W	AJS/AJkL	A-type indiaskvn body- II·« protein
Q255	OIL	hwriycharaewriied protein, atrivator oidweMrateDMlar signal-regulAted kin-.w pathway	(Jo
|lfr7	FIJL	Protein involved in inrracellwlarenveloped virion mstii rarion And cytoskeleton-dependent virion espen	(Jo	Inserivaaed derivative of DNA polymer»«, possibly oi hKterioptrage origin
|M>7	G5L	Fri*ion-e nt ry compky ?iib«n n	f-w
	H?R	fronein involved in MV* niemtimnebiogfnais
IMQ	AHL	hoiein involved in MV membrane biopnesk	f-w
1411	AI7L	fronein involved in MV membrane biojenesio
iMs	Lift	hoiein involved in MV membrane biojenesk	f-w
	liL	MV membfjne protein	(Jo
IMS	UL	Membrane protein eeoemial for vin*; entry	f-w
IMI	HR	Protein involved in MVfornurion. assembly ioniplei snbunin	fiS
Hit·		Prceein involved in MV iornwrion, assembly tompfercsiibonin	f<=5
1112	AWL	Protein involved in MV iornurion. assembly toniptarsnbunifl	fiS
IM1	A*	Prceein involved in MV morphogenesis	f<=5
Q2M	H3L	MV membrane protein involved inoeilafiadimem	(Jo
HI*	AH 51	MV membrane proteindiArenhanees vinden«	(Jo

^a NCVOG number.

^b Essentiality was determined for vaccinia virus. ^c MV, mature virion.

The late-stage genes of chordopoxviruses, as well as most intermediate and some early genes, contain a distinct sequence element within which transcription starts. This element 10 has the sequence TAAAT, where the second T usually corresponds to the second nucleotide of the translation initiation ATG codon of the respective gene. In the process of transcription initiation, the complement of this element serves as the template for the formation of the 5'-terminal poly(A) sequence that is present in many chordopoxvirus transcripts and is produced by RNA polymerase slippage. This TAAAT element is conserved in nearly all SGPV homologs of the respective chordopoxvirus genes (Table 5), suggesting that the main features of transcription initiation are shared by all chordopoxviruses.

Eight genes of SGPV have homologs in other NCLDVs and, thus, are formally assigned to NCVOGs, but as indicated by sequence similarity searches and phylogenetic analysis results (Table 4), they have probably been independently acquired by different viruses, which implies that they play important roles in virus-host interactions. Two of these genes (SGPV034 and SGPV139) encode RING finger proteins that could function as either specialized E3 subunits of ubiquitin ligases or inhibitors of ubiquitin pathways. RING finger-containing E3 proteins are encoded by many NCLDVs, including some of the orthopoxviruses, in which they are essential for pathogenicity. However, viral RING finger domains, including those encoded by the SGPV genome, show limited sequence similarity to each other and have probably been acquired independently. This independent acquisition was likely driven by the selection for virus interaction with the host ubiquitin networks. A similar trend of likely independent acquisition by diverse viruses is apparent for the DnaJ (J) domain, which was detected in the SGPV102 protein sequence. The J domain is present in mimiviruses, some phycodnaviruses, a single chordopoxvirus (molluscum contagiosum virus), as well as polyomaviruses. The polyomavirus J domain has been shown to function as a cochaperonin, enhancing the activity of the Hsc70 chaperone in the infected cells. A similar role in viral protein folding could be played by SGPV102.

Of special interest is the SGPV043 protein, a predicted serine/threonine protein kinase that is highly similar to the eukaryotic ribosomal protein S6 kinase (S6K), with which it shows up to 60% amino acid sequence identity. S6K is a component of the mTOR pathway and, more specifically, of the TORC1 complex, an environmental sensor that promotes anabolic pathways and inhibits catabolic pathways. Thus, this gene, which seems to have been convergently captured by SGPV and several other NCLDVs, could act as a regulator of the global metabolic state of virus-infected cells.

Only four SGPV genes appear to be unique acquisitions from cellular organisms, as they have no homologs in other NCLDVs. These are the metalloendopeptidase SGPV051, the thioredoxin SGPV147, the predicted DNA or RNA methyltransferase SGPV186, and the macrodomain-containing protein SGPV187, a putative O-acetyl-ADP-ribose deacetylase. Each of these proteins showed a high level of divergence from cellular homologs, presumably due to the high rate of evolution upon transfer to the viral genome, precluding a convincing inference of origin by phylogenetic analysis (not shown). The presence of the macrodomain is of special interest. Previously, this domain has been detected in several groups of animal positive-strand RNA viruses and has been shown to inhibit doublestrand RNA-dependent phosphorylation of the interferon regulatory factor 3 (IRF-3), a key transcription factor for interferon induction. The macrodomain of SGPV, to our knowledge, is the first domain of this family to be discovered in a DNA virus, and it might play a similar role as an inhibitor of the interferon pathway.

Conserved poxvirus genes that are missing in SGPV: distinct pathways of membrane biogenesis? As pointed out above, SGPV lacks numerous genes that are (nearly) fully conserved among the tetrapod-infecting chordopoxviruses, with the implication being that they are lost in SGPV (Table 6). Only three ancestral poxvirus genes appear to have been lost in SGPV; one of these, the gene for the protein phosphatase H1, was, apparently, also independently lost in some entomopoxviruses. The absence in SGPV of the A11, L2, A14, and A17 genes highlights a central functional theme that extends into the longer list of conserved chordopoxvirus genes that are missing in SGPV, namely, membrane biogenesis (Table 6). At least half of the missing genes (14 of 28) encode proteins implicated in this process. Of the seven subunits of a distinct protein complex involved in the association of the viroplasm with membranes, which is required for immature virion formation, only one, the protein kinase F10, an ancestral NCLDV protein that is likely to perform multiple functions, is represented by an ortholog in SGPV (Table 6; two complex subunits, D2 and A15, are not listed because they appear to have been lost in some other chordopoxviruses as well). Taken together, these findings imply that SGPV employs a pathway of membrane biosynthesis that is distinct from that of other chordopoxviruses. Several uncharacterized SGPV proteins contain predicted transmembrane segments (Table 5) but show no detectable sequence similarity to the sequences of pro-teins of other poxviruses shown to participate in membrane biogenesis; it appears likely that at least some of these SGPV proteins belong to the putative alternative pathway.

Among the other conspicuous gaps in the gene repertoire of SGPV are the singlestranded DNA-binding protein I3 and the DNA polymerase processivity factor A20, two proteins that are essential for VACV DNA replication. Among the predicted SGPV gene products, there are no obvious candidates that could replace these proteins, so the involvement of functionally analogous host proteins seems to be a distinct possibility.

Also missing in SGPV are two components of the thiol-disulfide oxidoreductase pathway, which is essential for the formation of the disulfide bonds in the subunits of the VACV fusion-entry complex, as well as envelope proteins L1 and F9. Orthologs of the L1 protein along with the upstream component of the thiol-disulfide oxidoreductase pathway, the E10 protein, are conserved in nearly all NCLDVs (38), with the implication being that the pathway as such is essential. The two missing subunits, the glutaredoxin G4 and the thioredoxin-like protein A2.5 have no orthologs in other NCLDV families either, indicating that the complete oxidoreductase pathway characterized in VACV evolved only after the divergence of SGPV and the rest of the chordopoxvi-ruses. The predicted SGPV thioredoxin (SGPV147) might be responsible, at least in part, for the missing portion of the pathway.

DISCUSSION

Poxvirus infection in salmon was suspected in the 1990s, as TEM showed apoptotic gill epithelial cells with poxvirus-like particles in samples submitted to the Norwegian Veterinary Institute from acute, high-mortality events in freshwater farms with juvenile fish. Typical poxvirus structures were further characterized in a TEM study of gill disease in Atlantic salmon (9), but no taxonomic assignment was possible in the absence of sequence data. In this study, we confirmed the presence of poxvirus particles and determined the sequence and phylogeny of salmon gill poxvirus, developed qPCR and IHC methods, and analyzed the disease from current as well as archival samples.

As there was no experimental model for SGPV disease, we obtained samples from fish with spontaneous cases of the suspect apoptotic gill disease in two hatcheries without other significant disease problems. As controls we included samples from fish involved in several gill disease outbreaks without apoptosis of gill epithelium as well as healthy fish. We found the SGPV infection by qPCR only in the disease cases and could link the SGPV infection in situ to the apoptotic respiratory epithelium by IHC. We found that the infection was widespread in the gills at least 3 days before the onset of severe clinical disease. Mortality coincided with blocking of the respiratory gill surfaces by two different mechanisms. The SGPV infection seems to induce massive apoptosis and detachment of the epithelium, resulting in the acute adherence of the thin gill lamellae. In other fish, an excessive proliferation of the epithelium blocked the respiratory surfaces. These findings indicate that viral replication precedes the gill pathologies that can be likened to atelectasis and solidification of the lungs, respectively. Hypoxia and osmoregulatory disturbances are the expected pathophysiological consequences from such lesions in fish. This is in keeping with the clinical experience that stopping feeding, raising oxygen levels, and avoiding all stress minimize mortality, which otherwise may approach 100% within hours in a tank of fish. Although the classical Koch's postulates remain to be fulfilled, our findings indicate that SGPV causes a distinct disease primarily affecting the gills in salmon. In fish, localized gill infection seems to be the rule for the suspected poxvirusesWith regard to systemic pathology and infection, we found that hemophagocytosis was associated with severe disease. In infectious salmon anemia, a severe orthomyxoviral disease of salmon, hemophagocytosis is due to virus attachment to the erythrocyte surfaces. However, only high or no CT values were found in organs of the poxvirus-infected salmon with hemophagocytosis, while low CT values were obtained from the gills in these fish. Hemophagocytosis is possibly a sign of circulatory disturbances aggravating the SGPV disease. It is noteworthy that lethargy, gill pathology, and hemophagocytosis are also reported in koi sleepy disease, associated with a pox virus of gills and seemingly not internal organs (10). Poxviruses are generally epitheliotropic, and skin tissue samples were positive by PCR both in this study and in the study of koi carp (10). However, we found no skin lesions in the salmon, and at present, we cannot exclude the possibility that skin tissue samples carry just virus shed from the gills. IHC seemed to indicate a very narrow cell tropism, since the simple, squamous lamellar epithelium of the gill was infected, while the adjacent stratified epithelium did not show signs of infection. The high mortality due to respiratory SGPV infection appears to be different from that in poxvirus infections in air-breathing vertebrates, where lung pathology is usually seen as part of a generalized infection. This could be related to the fundamental anatomical differences in the respiratory systems in the two vertebrate groups, where fish have their respiratory surface much more exposed to the exterior.

All salmon sampled a week after mortality had subsided in a tank were still infected, as shown by PCR. However, virus levels were generally lower and hemophagocytosis was much less prominent. These findings suggest that although recurrent, acute outbreaks in a tank are not reported by the farmers, SGPV infection may persist. For how long we do not know, and as the reservoir of infection is also unknown, we cannot rule out the possibility of reinfections. However, our archival samples do show that the infection is found not only in freshwater hatcheries but also in the seawater farms that receive the salmon for additional growth. In the cases of combined amoebic gill disease and SGPV disease that we confirmed here, 82% of the fish died. This is in agreement with the findings of other studies demonstrating that most gill diseases in the seawater rearing phase are complex, with multiple agents being present. Investigation of the role of SGPV in mixed infections will be an important future task, as the gill problems caused by these mixed infections cause considerable losses. To this end, we now have two new diagnostic methods, qPCR and IHC, for the detection of the SGPV in fish tissues, and these are useful for screening and resolving the complex pathology, respectively.

Judging from the archival samples, the SGPV disease emerged in the mid-1990s in Norwegian salmon farms over a wide geographical range. However, SGPV is distinct from other chordo-poxviruses that have been analyzed, and its reservoir is unknown. High mortality, like that caused by SGPV in salmon, can be a sign of a new host-agent system with low compatibility; on the other hand, intensive farming may have changed an old host-agent balance. Further studies are needed to clarify the reservoir and host range of SGPV and other aquatic poxviruses beyond salmon farming. The level of production of farmed food fish grew from 13 million to 66 million tons during the period between 1990 and 2012. Epidemics of orthomyxoviral disease virtually stopped salmon production in Chile and demonstrate the importance of disease control in fish farming. Also, for feral fish populations, introduction of new viruses may have serious consequences, as shown by the mass mortalities associated with rhabdoviral disease in the Great Lakes. We urgently need more knowledge of fish poxviruses, as the global trade and movement of aquaculture animals are growing. Research on the poxviruses of fishes may also bring cures for the aquaculture industry in the form of vaccines and the development of vectors.

Genome analysis of SGPV established the position of this fish-infecting virus at the base of the chordopoxvirus tree, as could be expected under the assumption of virus-host coevolution. However, the differences between the gene complements of SGPV and those of the rest of the chordopoxviruses are extensive, with SGPV lacking 38 genes otherwise conserved in chordopoxviruses. This difference in gene content is spread unevenly across the functional classes of viral genes. Most of the genes involved in genome replication and expression, as well as core and capsid structure and morphogenesis, are shared by SGPV and other chordopoxviruses. In sharp contrast, the majority of the chordopoxviruses genes implicated in viral membrane biogenesis are missing in SGPV. Chordopoxviruses employ a unique pathway of viral membrane derivation from the membranes of the endoplasmic reticulum of the infected cells that requires the participation of multiple viral proteins. The most parsimonious explanation for the absence of most of these proteins in SGPV is that this pathway, at least in its complete form, evolved in viruses infecting tetrapods; however, the alternative scenario, in which the pathway evolved in the ancestral chordopoxvirus but was subsequently lost in SGPV, cannot be ruled out. In addition, the multiprotein complex that in vaccinia virus is involved in viroplasm association with membranes, that is essential for virion maturation, and that so far appears to be conserved in all chordopoxviruses is missing in SGPV. Nevertheless, recognizable poxvirus crescent membranes, immature virions, and mature virions are formed. Thus, SGPV appears to employ a pathway of viral membrane biogenesis similar to that of other chordopoxviruses, despite the absence of key conserved proteins; multiple predicted membrane proteins of SGPV without homologs in other viruses could contribute to this alternative version of membrane biogenesis.

SGPV also lacks the proteins that are involved in the interaction of other chordopoxviruses with host defense systems, such as multiple paralogous genes that encode proteins containing kelch and ankyrin repeats as well as proteins involved in the suppression of host immune mechanisms. The conspicuous exception is the conserved B22R-like giant membrane proteins. However, the SGPV genome encodes numerous uncharacterized genes, many of which encode predicted membrane and secreted proteins as well as predicted nonglobular proteins with low-complexity sequences and simple repeats. Many, if not most, of the protein products of these genes are likely involved in currently unknown interactions with the immunity systems of the fish host. Experimental study of these uncharacterized proteins of SGPV could help the study of the pathogenesis of the gill disease in salmon and, more generally, the mechanisms of the poxvirus-host interaction.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Unless expressly described to the contrary, each of the preferred features described herein can be used in combination with any and all of the other herein described preferred features.

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Claims (16)

An isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 1-9 or a variant thereof which is at least 95% identical thereto over the total length of SEQ ID Nos: 1-9, and / or a nucleic acid sequence which is complementary to the nucleic acid sequence or variant thereof. A nucleic acid fragment of an isolated nucleic acid molecule according to SEQ ID NO: 2-9 or a variant thereof which is 95-100% identical thereto, the fragment comprising or consisting of at least 5 contiguous nucleic acid bases, the nucleic acid fragment being a nucleic acid primer capable of specifically to detect fish-pox virus in a sample. A nucleic acid fragment of an isolated nucleic acid molecule according to SEQ ID NO: 1 or a variant thereof which is 95-100% identical thereto, the fragment consisting of 20-100 contiguous nucleic acid bases of a nucleic acid molecule according to SEQ ID NO: 1, wherein the nucleic acid fragment is a nucleic acid primer , such as a primer as defined in any one of SEQ ID NOs: 8-9, which is capable of specifically detecting fishpox virus in a sample. A nucleic acid fragment of an isolated nucleic acid molecule as defined in claim 1, wherein the fragment consists of at least 5 contiguous nucleic acid bases of a nucleic acid molecule as defined in claim 1, wherein the nucleic acid fragment is a nucleic acid probe, such as a probe as defined in any NO of any : 4-7, which are capable of specifically detecting fishpox virus in a sample, the probe further comprising one or more markers for detecting the probe. A polypeptide encoded by a sequential strand of 48-1000 nucleic acid bases of a nucleic acid molecule as defined in claim 1, or a nucleic acid sequence which is inversely complementary thereto, such as a peptide according to any one of SEQ ID NO: 10-15. An antigen comprising or consisting of a polypeptide as defined in claim 5. The antibody specifically directed against the antigen of claim 6. Use of a nucleic acid fragment of a nucleic acid molecule according to SEQ ID NO: 1- 9 or a variant thereof which is at least 85% identical thereto, and / or a nucleic acid sequence which is complementary to the nucleic acid sequence or the variant thereof, the nucleic acid fragment consisting of at least 5 contiguous nucleic acid bases of the nucleic acid molecule or the variant thereof wherein the nucleic acid nucleus primer as defined in any one of SEQ ID NOs: 8-9, or a nucleic acid probe, such as a nucleic acid probe as defined in any one of SEQ ID NOs: 4-7, which is capable of specifically detecting fish smallpox virus in a sample, the probe optionally comprising one or more markers for detecting the probe, for specifically detecting the presence of smallpox virus, and / or diagnosing a smallpox virus infection, in a sample. Use of an antibody directed against an antigen consisting of a polypeptide encoded by a sequential strand of at least 12 nucleic acid bases of an isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 1-9 or a variant thereof which is at least 85% identical thereto, and / or a nucleic acid sequence complementary to the nucleic acid sequence or variant thereof, for specifically detecting the presence of smallpox virus, and / or diagnosing a smallpox virus infection, in a sample. Use of an isolated nucleic acid molecule as defined in claim 1 for the manufacture of a primer and / or probe which specifically detects fishpox virus. Use of an isolated nucleic acid molecule as defined in claim 1 or a nucleic acid fragment as defined in claim 2 for expressing a peptide, or a polypeptide as defined in claim 3, for the production of an antibody which specifically detects smallpox virus. A method of detecting a virus, such as smallpox virus, in a sample, such as a tissue sample from fish, the method comprising detecting at least 5 consecutive nucleic acid bases of a nucleic acid sequence according to any one of SEQ ID NOs: 1-9, or a variant which is at least 85% identical thereto, or a sequence which is complementary thereto. An ex vivo method, such as a method comprising the polymerase chain reaction method or in situ hybridization, for detecting the presence of a fishpox virus-specific nucleic acid, fishpox virus specific polypeptides or proteins, and / or fishpox virus, and / or diagnosing a fish-pox virus infection in a sample, the method comprising the steps of: a) contacting the sample with a nucleic acid fragment as defined in any one of claims 2-4, and / or an antibody as defined in claim 7; b) detecting the formation of a complex between a fishpox virus-specific nucleic acid or polypeptide, respectively, and the nucleic acid fragment or antibody, respectively; wherein the presence of a complex indicates the presence of a fishpox virus-specific nucleic acid, a fishpox virus-specific polypeptide or protein, and / or fishpox virus, and / or a fishpox virus infection in the sample. A diagnostic kit for diagnosing a viral infection, such as smallpox virus infection, in an individual, which kit comprises one or more of an isolated nucleic acid molecule as defined in claim 1, a nucleic acid fragment as defined in any one of claims 2-4, a polypeptide as defined in claim 5, an antigen as defined in claim 6, and / or an antibody as defined in claim 7 and reagents for performing a diagnosis, and optionally instructions for performing such a diagnosis. A pharmaceutical composition comprising one or more of an isolated nucleic acid molecule as defined in claim 1, a nucleic acid fragment as defined in any one of claims 2-4, a polypeptide as defined in claim 5, an antigen as defined in claim 6, and / or an antibody as defined in claim 7. 16. Et an isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 1-9 or a variant thereof which is at least 85% identical thereto, and / or a nucleic acid sequence which is complementary to the nucleic acid sequence or the variant thereof; an isolated nucleic acid fragment as defined in claim 8; a polypeptide encoded by a sequential strand of at least 12 nucleic acid bases of a nucleic acid molecule according to SEQ ID NO: 1-9 or a variant thereof which is at least 85% identical thereto, and / or a nucleic acid sequence which is complementary to the nucleic acid sequence or the variant thereof, or a nucleic acid sequence which is inversely complementary thereto, such as a peptide according to any one of SEQ ID NO: 10-15; an antigen consisting of a polypeptide encoded by a sequential strand of at least 12 nucleic acid bases of an isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 1-9 or a variant thereof which is at least 85% identical thereto, and / or a nucleic acid sequence complementary to the nucleic acid sequence or variant thereof; and or an antibody as defined in claim 7 for use in the prevention and / or treatment of a smallpox virus infection.