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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/12011—Astroviridae
  • C12N2770/12021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/12011—Astroviridae
  • C12N2770/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/12011—Astroviridae
  • C12N2770/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to a novel type of porcine astroviruses and the uses thereof.
• the invention also relates to parts and subunits and variants of said viruses, compositions comprising the same, their manufacture, and their uses e.g., for the design and production of vaccines for treating pigs.
• the invention also concerns methods for treating and/or preventing astrovirus-associated diseases, as well as methods and materials for detecting an infection.
• Astroviruses are small, non-enveloped viruses with single stranded 6.2-7.8 kb-long R A genome of positive polarity.
• the family Astroviridae is currently divided into two genera: the genus Mamastrovirus of mammal-infecting viruses and the genus Avastrovirus of avian viruses.
• the genetically heterogenic astroviruses that are widespread among mammals and avian species are generally associated with gastroenteritis.
• porcine astroviruses (PoAstVs) were identified mainly from diarrhoeic faecal samples, although the etiological role of astrovirus infection in gastroenteritis or in other diseases among swine is not settled.
• the applicant has succeeded in detecting, obtaining and characterizing new astrovirus strains from brain and spinal cord samples obtained from diseased pigs in Europe. These viruses were detected from samples of pigs with neurological disorders, but not in healthy pigs. The virus was isolated from pigs showing clinical signs of posterior leg weakness/paraplegia, pitching (Stage 1),; paralysis of both legs, skin pain (Stage 2), loss of consciousness, paresis and serious spastic paralysis of muscles (end stage, Stage 3) with high mortality rate among newly weaned (25-35-day-old) pigs. Affected pigs that were necropsied had encephalomyelitis and neural necrosis.
• the four strains were identified by RT-PCR and in situ hybridization (ISH) in brain and spinal cord samples in 6 cases and referred to as 'TNfeuroinvasive porcine astroviruses".
• ISH in situ hybridization
• the highest viral loads were present in brainstem and spinal cord.
• Neuroinvasive porcine astroviruses were also detected in archived, formalin fixed paraffin embedded (FFPE) brain and spinal cord samples from other affected pigs and/or from other farms.
• Viral RNA was predominantly restricted to neurons, particularly in the brainstem, cortex, cerebellum (Purkinje cells), and all parts of the spinal cord.
• the virus was generally undetectable from faeces but present in respiratory samples indicating a possible respiratory infection.
• the virus was also detectable in other internal organs/tissues such as sera, lung, tonsils, hearth, submandibular lymph node and salivary glands
• the applicant has sequenced the complete genomes of three strains (KY073229, KY073231 and KY073232) and the complete ORF2 of a fourth strain (KY073230).
• the strains exhibit a strong homology with each other at the nucleotide level, typically exceeding 99%, and much lower with previously known, essentially gastroenteric, astro virus strains, typically below 91 >.
• the new strains can thus be considered as being representative of a new clade of porcine astro viruses, designated in the present as neuroinvasive porcine type-3 astroviruses (NI-PoAstV-3).
• the invention further discloses the sequence of proteins encoded by these viruses, including the capsid protein (ORF2) and well as regulatory proteins ORFla and ORFlab.
• ORF2 capsid protein
• ORFla regulatory proteins
• ORFlab regulatory proteins
• the invention discloses a very high level of identity between the ORF proteins of this new clade of viruses, as well as distinctive sequence variations as compared to other astrovirus capsid proteins.
• the invention thus reports isolation and characterization of a novel class of neuroinvasive porcine astroviruses and provides novel methods, reagents and compositions for treating, preventing or detecting such infections and related diseases. Summary
• An object of the invention relates to isolated neuroinvasive porcine type-3 astroviruses, as well as parts, subunits and variants thereof.
• the viruses may be native, attenuated, or inactivated (e.g. killed).
• Such viruses refer to a clade of viruses having at least 91%> nucleotide sequence identity over the entire genome to any of KY073229, KY073230, KY073231 or KY073232, preferably at least 95%, more preferably at least 99%, and being preferably neuroinvasive in swine.
• the invention particularly relates to an isolated porcine astrovirus comprising:
• an ORF2 nucleic acid sequence encoding an ORF2 polypeptide comprising or consisting of the amino acid sequence of anyone of SEQ ID NOs: 5 to 8 or the complementary strand thereof, and/or
• an ORFla nucleic acid sequence encoding an ORFla polypeptide comprising or consisting of the amino acid sequence of anyone of SEQ ID NOs: 9 to 11 or the complementary strand thereof, and/or (f) an ORFlab nucleic acid sequence selected from nucleotide residues 31 to 4085 of anyone of SEQ ID NOs: 1 to 3 or the complementary strand thereof, and/or
• an ORFlb nucleic acid sequence encoding an ORFlb polypeptide comprising or consisting of the amino acid sequence of anyone of SEQ ID NOs: 15 to 17 or the complementary strand thereof, or
• the invention particularly relates to isolated porcine type-3 astro viruses comprising:
• viruses are neuroinvasive in swine.
• the invention also relates to isolated porcine type-3 astroviruses comprising an ORF2 nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astroviruses comprising an ORF2 nucleic acid sequence selected from:
• nucleic acid sequence encoding an ORF2 polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8, or the complementary strand thereof, or (b) a nucleotide sequence having a homology of at least 87%, at least 88%, at least89, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%>, at least 97%, at least 98%>, or at least 99% to a nucleotide sequence of (a), or
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFla nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFla nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFlab nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFlab nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFlb nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFlb nucleic acid sequence selected from:
• a further object of the invention is a host cell containing a virus as defined above.
• Another object of the invention relates to a method of producing a porcine astrovirus, comprising seeding a competent host cell with a seed of culture of a virus as defined above, culturing or growing the cells under suitable conditions in a manner allowing production of the virus, and optionally harvesting the porcine astrovirus from said cells.
• the invention also relates to a nucleic acid molecule comprising:
• nucleotide sequence comprising or consisting of nucleotide residues 4075 to 6339 of anyone of SEQ ID NOs: 1 to 3, or nucleotide residues 31 to 2559 of anyone of SEQ ID NOs: 1 to 3, or nucleotide residues 31 to 4085 of anyone of SEQ ID NOs: 1 to 3, or nucleotide residues 2559 to 4085 of anyone of SEQ ID NOs: 1 to 3, or the complementary strand thereof, or
• nucleic acid sequence encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or the complementary strand thereof, or (d) a nucleotide sequence having a homology of at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%>, at least 97%, at least 98%>, or at least 99% to a nucleotide sequence of (a), (b) or (c), or
• the invention also relates to polypeptides (e.g., proteins or peptides) encoded by or derived from such nucleic acid molecules.
• a particular object of the invention is a polypeptide selected from:
• a further object of the invention resides in a vector, recombinant virus, plasmid or host cell comprising a nucleic acid molecule as defined above or encoding a polypeptide as defined above.
• a further object of the invention is a composition or vaccine comprising a virus, a subunit thereof, a polypeptide, a nucleic acid, vector, or host cell as defined above, and a veterinary acceptable vehicle or excipient.
• the invention also relates to a composition or vaccine as defined above for use for treating and/or preventing a porcine astrovirus infection or associated disease, or for inducing or stimulating an immune response against porcine astrovirus in a mammal.
• the invention also provides a method of inducing or stimulating an immune response against porcine astrovirus in a mammal, comprising administering to the mammal an effective amount of a composition or vaccine as defined above.
• the invention further relates to a method of treating and/or preventing a porcine astrovirus-associated disease in an animal comprising administering to said animal an effective amount of a composition or vaccine as defined above.
• the invention also provides a method of treating and/or preventing encephalomyelitis or paralysis in an animal, comprising administering to said animal an effective amount of a composition or vaccine as defined above.
• a further object of the invention is a container or kit comprising an effective amount a composition or vaccine as defined above.
• the kit comprises the container and an instruction manual including information for the administration of the composition or vaccine into pigs for treating and/or preventing porcine astrovirus- associated diseases.
• the invention also discloses and provides diagnostic kits or reagents comprising nucleic acid probes or primers or antibodies or ligands that bind to a NI-PoAst3 virus or polypeptide.
• Preferred primers or probes comprise a fragment of a nucleotide sequence as defined above.
• the invention particularly relates to a polyclonal or monoclonal antibody, or a variant thereof, which is capable of binding to a virus or cell or polypeptide of the invention, or sub-units thereof.
• the antibody preferentially binds to a virus or cell or polypeptide of the invention essentially without binding a type 1 or type 2 astrovirus, or essentially without binding a type 3 astrovirus of a different clade.
• the invention also relates to a method of diagnosing the presence of a virus in an animal comprising contacting a tissue or fluid sample with the diagnosing reagent as defined above, and detecting the presence of a virus.
• Figure 1 Genome map of the neuroinvasive porcine astrovirus strain NI-Brain/9- 2016a/HUN (KY073229) together with the location of RT-PCR product used for different astrovirus screening reactions and RT-qPCR analyses.
• the black arrow indicates the localization of a ribosomal frame-shift during the synthesis of ORFlab peptide.
• Figure 2 Logarithmic graph of the viral copy numbers of porcine astrovirus 3 in different organs determined by SYBR Green based RT-qPCR.
• BS brainstem
• SC- C/T/L cervical-, thoracic-, or lumbar spinal cord
• NM nasal mucosa
• SG salivary gland
• CNS central nervous system.
• the analyzed samples were the same as in Table 1 and originated from five different animals with the IDs found in the top right corner of the picture. All the samples which were porcine astrovirus 3 positive only by nested RT- PCR were found negative by qPCR.
• FIG. 3 Tissue sections of cervical spinal cord (A), brainstem (B, C) and cerebellum (D) stained with hematoxylin and eosin from a pig shows the signs of end- stage encephalomyelitis (Stage 3).
• the mononuclear perivascular cuffs with vasculitis (black arrowheads) together with significant neuronal necrosis (white arrowheads), neurophagia (white double arrowheads) and multifocal microgliosis are present in all sections.
• the black arrows in section D indicate the signs of meningitis. Scale bars: A, D: 50 ⁇ , Insert A, B, C: 20 ⁇ .
• FIG 4 Sections of the cervical spinal cord (A-E), cerebellum (F-J), cortex (L, M) from the index animal (GD-1) and brainstem (K) from an additional affected Stage 1 animal (GD-7).
• Hematoxylin and eosin A, D, F, I, L.
• Gliosis black arrows is multifocal within the gray matter (A,D) and in the molecular layers (F, I, L and M).
• Neuronal degeneration and necrosis is evident by hypereosinophilia, angular degeneration, loss of neuronal detail, and vacuo lation (double arrows) (A, D).
• ISH neuroinvasive porcine astrovirus: B, E, G, J, K, M.
• Hybridization of the neuroinvasive porcine astrovirus probe is restricted to neurons (white arrowheads, B, E, K) or limited to Purkinje neurons (double black arrowheads, G, J) with extension into dendritic processes that course through the molecular layer (black arrowheads G, J).
• Hybridization of the porcine astrovirus 3 probe black arrowhead, M
• C, H Using a control probe on a serial section, no hybridization is detectable.
• DapB probe Scale bars: A-C, F-H,: 500 ⁇ ; D,E, I-M: 50 ⁇ .
• the invention defines a novel clade of astroviruses and provides means, compositions and methods for producing such viruses, subunits thereof, material derived therefrom, as well as detecting, preventing and treating infections associated with such viruses in mammals, particularly in pigs.
• the invention relates to any neuroinvasive porcine astrovirus capable of being isolated from a nervous tissue sample of a pig, especially brain or spinal cord.
• the present invention more particularly relates to purified or isolated preparations of NI-PoAstV-3 strains, the sequences of which were deposited at Genbank under the following numbers, as well as any neuroinvasive porcine astroviruses having a significant serological and/or structural similarity with these strains.
• the sequences of the deposited strains are detailed below:
• Strain NI-Brain/173-2016a/HUN, Genbank No. KY073231 SEQ ID NOs: 2 (genome), 7 (ORF2 protein), 10 (ORFla protein), 13 (ORFlab protein) and 16 (ORFlb protein); Strain NI-Brain/386-2015/HUN, Genbank No. KY073232: SEQ ID NOs: 3 (genome), 8 (ORF2 protein), 11 (ORFla protein), 14 (ORFlab protein) and 17 (ORFlb protein).
• the invention particularly relates to isolated porcine type-3 astroviruses comprising
• viruses represent a novel clade of astroviruses, which may be defined by a level of sequence identity above 91%>, preferably above 95%, even more preferably above 98%.
• the astroviruses of the invention comprise SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3, or the complementary strand thereof, or a nucleotide sequence having at least 98%> sequence identity therewith.
• the applicant has further analyzed the nucleotide sequences of these viruses and identified open reading frames encoding proteins ORF2, ORFla, ORFlb and ORFlab. These ORFs are located at nucleotide positions 31 to 4085 (ORFlab), 31 to 2559 (ORFla), 2559 to 4085 (ORFlb) and 4075 to 6339 (ORF2).
• ORFlab open reading frames encoding proteins
• ORFla ORFla
• ORFlb ORFlb
• ORF2 open reading frames encoding proteins
• the invention also relates to isolated porcine type-3 astroviruses comprising an ORF2 nucleic acid sequence selected from:
• nucleic acid sequence encoding an ORF2 polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8, or the complementary strand thereof, or
• the invention relates to any isolated porcine astro virus comprising an ORF2 nucleic acid sequence selected from an ORF2 polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8, the complementary strand thereof, or a nucleotide sequence having a homology of at least 97%, at least 98%, or at least 99% thereto.
• the ORF2 nucleic acid has at least 87% identity with nucleotide residues 4075 to 6339 of anyone of SEQ ID NOs: 1 to 3, or with SEQ ID NO: 4, or the complementary strand thereof, and encodes at least one of the amino acid residues listed in table 3 (the position being given by reference to SEQ ID NO: 5, 6, 7 or 8), e.g., R29, S34, R38, V55, T57, T61, L439, S453, F457, Y559, A570, N572, D581, 1601, S617, T628, S678, and/or 1696.
• the invention also relates to isolated porcine type-3 astroviruses comprising an ORFla nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFla nucleic acid sequence selected from:
• the invention relates to any isolated porcine astro virus comprising an ORFla nucleic acid sequence selected from an ORFla polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, the complementary strand thereof, or a nucleotide sequence having a homology of at least 97%, at least 98%, or at least 99% thereto.
• the invention also relates to isolated porcine type-3 astroviruses comprising an ORFlab nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astroviruses comprising an ORFlab nucleic acid sequence selected from:
• the invention relates to any isolated porcine astro virus comprising an ORFlab nucleic acid sequence selected from an ORFlab polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14, the complementary strand thereof, or a nucleotide sequence having a homology of at least 97%, at least 98%, or at least 99% thereto.
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFlb nucleic acid sequence selected from:
• the invention also relates to isolated porcine type-3 astro viruses comprising an ORFlb nucleic acid sequence selected from:
• the invention relates to any isolated porcine astro virus comprising an ORFlab nucleic acid sequence selected from an ORFlb polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17, the complementary strand thereof, or a nucleotide sequence having a homology of at least 97%, at least 98%, or at least 99% thereto.
• the viruses of the invention may be isolated from a physiological sample or from a tissue sample, and/or propagated or maintained on cell lines such as especially pig kidney or testis cells or cell lines, e.g., for their multiplication. They may also be produced by seeding a competent cell with a virus stock or a nucleic acid molecule containing a genome of such a virus, as defined in the present invention. In a particular embodiment, a virus is prepared by infecting a competent cell with a vector containing a nucleic acid genome of the virus and collecting the virus produced.
• the NI-PoAstV-3 of the invention are typically provided in an isolated form.
• isolated is meant to refer to a virus strain which is not in a native environment of a wild-type virus.
• the virus may be a component of a cell culture or other artificial medium where it can be propagated; a component or a cell culture supernatant; a component of a pharmaceutical composition; or partially or completely purified from its native environment.
• the viruses may be isolated or purified using techniques known per se in the art such as centrifugation, affinity purification, gel permeation techniques, chromatographic methods, and the like.
• the degree of homology between two nucleic acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443- 453).
• GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5. 0 and GAP extension penalty of 0.3.
• Nucleic acid molecules may be aligned to each other using the Pileup alignment software, available as part of the GCG program package, using, for instance, the default settings of gap creation penalty of 5 and gap width penalty of 0.3.
• the invention relates to NI-PoAstV-3 viruses in attenuated or inactivated (i.e., killed) form.
• the terms "killed” or “inactivated” refer to a significant or complete reduction in the infectivity of the virus(es).
• an inactivated virus is a virus which has been structurally altered and is unable to infect a cell susceptible to infection by a non- inactivated virus.
• the term "attenuated” refers to a substantial reduction of pathogenicity, typically at least 50% reduction, more preferably at least 80% reduction.
• an attenuated virus can replicate and/or infect a cell, but essentially does not cause a disease in a mammal.
• inactivation may be carried out by exposing the virus to a chemical agent such as formaldehyde, paraformaldehyde, ⁇ -propiolactone, ethyleneimine, binary ethyleneimine (BEI), thimerosal, or derivatives thereof.
• a chemical agent such as formaldehyde, paraformaldehyde, ⁇ -propiolactone, ethyleneimine, binary ethyleneimine (BEI), thimerosal, or derivatives thereof.
• inactivation may be carried out by physical treatments such as heat treatment or sonication. Methods of inactivation are well known to those of skill in the art.
• the inactivated virus may be concentrated by conventional concentration techniques, in particular by ultrafiltration, and/or purified by conventional purification means, in particular using chromatography techniques including but not limited to gel- filtration, ultracentrifugation on a sucrose gradient, or selective precipitations, in particular in PEG.
• Methods for attenuating a virus are also known per se in the art and comprise, e.g., the passage on cells, preferably passages on pig cells, especially lines, such as PK/15 cells (for example from 50 to 150, especially of the order of 100, passages).
• the present invention relates to a nucleic acid molecule comprising a nucleotide sequence of all or part of the genome of a NI-PoAstV-3 virus, or a variant thereof.
• the nucleic acid molecule may comprise a sequence of an entire genome, or variants thereof.
• the nucleic acid may comprise a sequence of an ORF, or a fragment or variant thereof.
• the nucleic acid may comprise a regulatory sequence.
• the nucleic acid molecule comprises:
• nucleotide sequence comprising or consisting of nucleotide residues 4075 to 6339 of anyone of SEQ ID NOs: 1 to 3, or nucleotide residues 31 to 2559 of anyone of SEQ ID NOs: 1 to 3, or nucleotide residues 31 to 4085 of anyone of SEQ ID NOs: 1 to 3, or nucleotide residues 2559 to 4085 of anyone of SEQ ID NOs: 1 to 3, or the complementary strand thereof, or
• nucleic acid sequence encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 1 1 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or the complementary strand thereof, or
• the nucleotide sequence in (d) has a homology of at least 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% with the reference nucleotide sequence.
• the fragment in (f) is a fragment of at least 15, at least 20, or at least 25 consecutive nucleotides of the reference nucleotide sequence, more preferably a fragment of less than 80 nucleotides, most preferably a fragment of between 20 and 70 consecutive nucleotides of the reference sequence, particularly of between 25 and 60.
• the nucleic acid molecule may be double-stranded or single- stranded DNA or RNA, as well as analogues thereof such as those containing modified backbones.
• Single- stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
• the nucleic acid molecules according to the invention may be provided in the form of a nucleic acid molecule per se such as naked nucleic acid molecules; or incorporated in a vector, virus or host cell, either from prokaryotic or eukaryotic origin.
• Vectors include cloning and/or expression vectors that contain a nucleic acid molecule of the invention.
• the vectors of the present invention may, for example, comprise a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.
• the invention relates to a vector comprising a nucleotide sequence encoding an ORF2 polypeptide as defined in the present invention.
• the invention relates to a vector comprising a nucleotide sequence encoding a fragment of an ORF2 polypeptide as defined in the present invention. In another particular embodiment, the invention relates to a vector comprising a nucleotide sequence encoding an ORFla polypeptide as defined in the present invention.
• the invention relates to a vector comprising a nucleotide sequence encoding an ORFlab polypeptide as defined in the present invention.
• the vector may be a plasmid, a cosmid, an artificial chromosome, a recombinant virus, and the like.
• E. coli or a baculovirus will be preferably used (U.S. Pat. No. 4,745,051).
• the coding sequence(s) or their fragments are integrated into the baculovirus genome (e.g. the baculovirus Autographa californica Nuclear Polyhedrosis Virus AcNPV) and the latter is then propagated on insect cells, e.g. Spodoptera frugiperda Sf9 (deposit ATCC CRL 1711).
• the subunits can also be produced in eukaryotic cells such as yeasts (e.g. Saccharomyces cerevisiae) or mammalian cells (e.g. CHO, BHK).
• the vector may be a plasmid or a recombinant virus, such as a recombinant Swinepox virus or a recombinant adenovirus, for instance, or a baculovirus.
• the invention provides a host cell transformed with a nucleic acid molecule of the invention as defined above.
• host cells include eukaryotic and prokaryotic cells.
• eukaryotic cells include mammalian (e.g., pig), fungal (e.g. Saccharomyces cerevisiae), insect and plant cells.
• Prokaryotic cells include, for example, bacteria such as E. coli.
• Such cells may be maintained or cultivated in any suitable medium. They may be frozen or lyophilized, for storage or maintenance.
• the invention also relates to polypeptides (or proteins) encoded by or derived from a virus or a nucleic acid molecule of the invention, as well as variants and fragments thereof.
• Polypeptides (including proteins or peptides) of the invention are preferably in isolated (or purified form). They may be isolated or obtained by purification from natural sources, or obtained by recombinant techniques or by chemical synthesis, or combinations thereof. When produced by chemical synthesis, they can contain unnatural amino acids. They may be further modified, e.g., to improve their stability or activity.
• the invention relates to a polypeptide encoded by ORFla, ORFlb, ORFlab, or ORF2 of a NI-PoAstV-3 of the invention, or variants or fragments thereof.
• a particular object of the invention is a polypeptide selected from:
• polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 1 1 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17,
• polypeptide fragment designates typically a polypeptide containing at least 5 and up to 100 consecutive amino acids, preferably between 10 and 80 consecutive amino acids, even more preferably between 10 and 50, or between 10 and 30 consecutive amino acids.
• Preferred fragments are immunogenic, i.e., are able to induce an immune response (preferably an anti-NI-PoAstV-3 immune response) when administered to a mammal.
• preferred fragments contain an epitope, which may be a T-cell or a B-cell epitope.
• polypeptide fragments of the invention are represented by polypeptides comprising, or consisting essentially of, amino acid residues 1 to 10, 15- 25, 30-50, 55 to 80 of anyone of SEQ ID NOs: 5 to 8.
• Preferred polypeptides under (b) above include polypeptides having at least 97%, at least 98%o, or at least 99% sequence identity to a polypeptide of (a).
• Such polypeptides typically contain one or more sequence variations as compared to the reference polypeptide, such as one or more amino acid deletion, substitution, or addition.
• one or more amino acids may be replaced by "conservative" amino acids, i.e., amino acids which upon substitution do not substantially alter a biological or immunological property of the polypeptide.
• conservative substitutions may include the replacement, for example, of leucine by valine or isoleucine, the replacement of aspartic acid by glutamic acid, or the replacement of arginine by lysine.
• a polypeptide of the invention is a polypeptide comprising or consisting essentially of an amino acid sequence SEQ ID NOs: 5, 6, 7 or 8, or which has at least 90%>, preferably at least 95% sequence identity with SEQ ID NOs: 5, 6, 7 or 8, or a fragment of at least 10 consecutive amino acids thereof.
• a polypeptide of the invention is a polypeptide comprising or consisting essentially of amino acid sequence SEQ ID NO: 5 or a sequence having at least 87%, preferably at least 97% identity to SEQ ID NO: 5, or a fragment of at least 10 consecutive amino acids thereof, preferably at least 97%.
• a polypeptide of the invention is a polypeptide comprising or consisting essentially of amino acid sequence SEQ ID NO: 6 or a sequence having at least 87%, preferably at least 97% identity to SEQ ID NO: 6, or a fragment of at least 10 consecutive amino acids thereof.
• a polypeptide of the invention is a polypeptide comprising or consisting essentially of amino acid sequence SEQ ID NO: 7 or a sequence having at least 87%, preferably at least 97% identity to SEQ ID NO: 7, or a fragment of at least 10 consecutive amino acids thereof.
• a polypeptide of the invention is a polypeptide comprising or consisting essentially of amino acid sequence SEQ ID NO: 8 or a sequence having at least 87%, preferably at least 97% identity to SEQ ID NO: 8, or a fragment of at least 10 consecutive amino acids thereof.
• Preferred ORF2 polypeptides of the invention contain at least one of the following amino acids (the position being given by reference to SEQ ID NO: 5, 6, 7 or 8): R29, S34, R38, V55, T57, T61, L439, S453, F457, Y559, A570, N572, D581, 1601, S617, T628, S678, and/or 1696.
• polypeptides according to the invention polypeptides that are recognized by antibodies produced during infection by NI-PoAstV-3 are particularly advantageous, as well as polypeptides which can induce or stimulate anti-NI-PoAstV-3 antibodies.
• polypeptides may be further modified, e.g., to improve or modify their activity, immunogenicity, stability, etc.
• the polypeptides may be glycosylated. They may be modified (or designed) to include non-natural amino acids or non-peptide bonds. They may also be modified by lateral or terminal groups to increase their resistance to proteases.
• compositions and vaccines are provided.
• compositions such as vaccines, comprising a virus, nucleic acid, vector, polypeptide or cell as defined above.
• compositions and vaccines include an agent which may be used to stimulate the immune system of an animal against a pathogen.
• the compositions and vaccines may comprise a whole NI-PoAstV-3 virus, in native (live), attenuated, or inactivated (e.g., killed) form, a subunit or a portion thereof, a recombinant vector containing an insert encoding an immunogenic polypeptide thereof, a fragment of DNA capable of inducing an immune response, a NI-PoAstV-3 polypeptide, a cell containing such elements, or a combination thereof.
• Such compositions and vaccines can be prepared according to standard techniques well known to those skilled in the art.
• the composition or vaccine comprises an inactivated NI-PoAstV- 3 virus.
• inactivated viruses may be obtained by treating the virus with inactivating agents such as formalin or acids, or by irradiation with ultraviolet light or X-rays, for instance.
• the composition or vaccine comprises an attenuated NI- PoAstV-3 virus.
• attenuated viruses may be obtained by serial passage through cell cultures, or by gene deletions for instance.
• the composition or vaccine comprises a NI-PoAstV-3 subunit.
• Such subunit vaccines typically contain an antigenic polypeptide of a NI-PoAstV-3, e.g., a protein encoded by a NI-PoAstV-3 virus, or a fragment thereof, or a variant thereof.
• a capsid protein such as the protein encoded by the ORF2 is employed as the antigenic component of the subunit vaccine, or a fragment or variant thereof.
• the composition or vaccine comprises a vector encoding a NI- PoAstV-3 antigen.
• vectorized vaccines typically contain recombinant plasmid or virus containing a nucleic acid sequence encoding an antigenic polypeptide of a NI- PoAstV-3, e.g., a protein encoded by a NI-PoAstV-3 virus, or a fragment thereof, or a variant thereof.
• the antigenic polypeptide is a capsid protein, such as the protein encoded by the ORF2, or a fragment or variant thereof.
• the vector may be a recombinant virus such as a baculovirus, or a swinepox virus, or a circovirus.
• Vaccines of the invention typically comprise an immunologically effective amount of a NI-PoAstV-3 virus or antigenic material in a pharmaceutically or veterinary acceptable vehicle.
• pigs become at least partially or completely immune to NI-PoAstV-3 infections, or resistant to developing moderate or severe NI- PoAstV-3 infections.
• NI-PoAstV-3 vaccines may be used to elicit a humoral and/or a cellular response.
• the amount required for an immunologically effective dose may vary depending on factors such as the age and general condition of the subject, the nature of the formulation and the mode of administration. Appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. For instance, methods are known in the art for determining or titrating suitable dosages of an astrovirus vaccine to find minimal effective dosages based on the weight of the non human animal subject, concentration of the vaccine and other typical factors.
• the dosage of the vaccine, concentration of components therein and timing of administering the vaccine, which elicit a suitable immune response can be determined by methods such as by antibody titrations of sera, e ⁇ g., by ELISA and/or seroneutralization assay analysis and/or by vaccination challenge evaluation.
• vaccines according to the present invention comprise from 10 3 to 10 10 TCIDso NI-PoAstV-3 viral particles per mL. In another particular embodiment, vaccines according to the present invention comprise from 0.001 ⁇ g to lg of a NI-PoAstV-3 polypeptide per mL. The polypeptide is typically an ORF2 polypeptide or a fragment thereof In another particular embodiment, vaccines according to the present invention comprise from 0.001 ⁇ g to lg of a NI-PoAstV-3 nucleic acid per mL.
• the nucleic acid is typically a vector containing a sequence encoding a NI-PoAstV-3 ORF2 protein or a fragment thereof
• Compositions and vaccines may comprise other ingredients, well known by one of ordinary skill in the art, such as pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze drying stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives, depending on the route of administration.
• Examples of pharmaceutically acceptable carriers, excipients or diluents include, but are not limited to demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, arachis oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as light liquid paraffin oil, or heavy liquid paraffin oil; squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, carboxymethylcellulose sodium salt, or hydroxypropyl methylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol
• the carrier or carriers will form from 10% to 99.9% by weight of the vaccine composition and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.
• adjuvants include, but are not limited to, aluminium hydroxide (alum), immunostimulating complexes, non-ionic block polymers or copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-a, IFN- ⁇ , IFN-y, etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP) and the like.
• alum aluminium hydroxide
• immunostimulating complexes include, but are not limited to, aluminium hydroxide (alum), immunostimulating complexes, non-ionic block polymers or copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-a, IFN- ⁇ , IFN-y, etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP) and the like.
• MDA monophosphoryl lipid A
• MDP muramyl dipeptide
• Suitable adjuvants include, for example, aluminium potassium sulphate, heat-labile or heat-stable enterotoxin isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc.
• Toxin-based adjuvants such as diphtheria toxin, tetanus toxin and pertussis toxin may be inactivated prior to use, for example, by treatment with formaldehyde.
• freeze-drying stabilizer may be for example carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, and derivatives thereof.
• Vaccines may additionally comprise at least one immunogen from at least one additional pathogen, e ⁇ g., a pig pathogen such as Actinobacillus pleuropneunomia; Adenovirus; Alphavirus such as Eastern equine encephalomyelitis viruses; Balantidium coli; Bordetella bronchiseptica; Brachyspira spp., preferably B. hyodyentheriae, B.pilosicoli, B. innocens, Brucella suis, preferably biovars 1,2 and 3; Classical swine fever virus, African swine fever virus; Chlamydia and Chlamydophila sp. and preferably C. pecorum and C.
• additional pathogen e ⁇ g., a pig pathogen such as Actinobacillus pleuropneunomia
• Adenovirus such as Eastern equine encephalomyelitis viruses
• Balantidium coli Bordetella
• Circovirus preferably PCV1 or PCV2
• Clostridium spp. preferably CI. difficile, CI. perfringens types A,B and C, Cl.novyi, Cl.septicum, Cl.tetani; Digestive and respiratory Coronavirus; Cryptosporidium parvum ; Eimeria spp; Eperythrozoonis suis currently named Mycoplasma haemosuis; Erysipelothrix rhusiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtypes 1,7 and 14; Hemagglutinating encephalomyelitis virus; Isospora suis ; Japanese Encephalitis virus; Lawsonia intracellularis; Leptospira spp., preferably Leptospira australis, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemor
• M. avium, M. intracellulare and M.bovis Mycoplasma hyponeumoniae; Parvovirus ; Pasteurella multocida; Porcine cytomegolo virus; Porcine parovirus, Porcine reproductive and respiratory syndrome virus: Pseudorabies virus; Rotavirus; Sagiyama virus ; Salmonella spp. preferably, S. thyhimurium and S.choleraesuis; Staphylococcus spp. preferably, S.
• Streptococcus spp. preferably Strep, suis; Swine cytomegalovirus ; Swine herpes virus; Swine influenza virus; Swine pox virus; Toxoplasma gondii ; Vesicular stomatitis virus and virus of exanthema of swine; or other isolates and subtypes of porcine circovirus.
• Vaccines may be liquid formulations such as an aqueous solution, water-in-oil or oil-in- water emulsion, or water-in-oil-in-water emulsion, syrup, an elixir, a tincture, a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions.
• Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems.
• Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents.
• Preferred PCV2 vaccines comprise inactivated PCV2B-Rm according to the invention in the forms of emulsions, for example water-in-oil or oil-in- water, which may be prepared according to well-known techniques in the art.
• the route of administration can be percutaneous, via mucosal administration, or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal).
• Vaccines compositions according to the present invention may be administered alone, or can be co-administered or sequentially administered with other treatments or therapies.
• the present invention provides a method of immunizing or inducing immune response in mammals, particularly in pigs, comprising administering a virus, nucleic acid, polypeptide, host cell, composition or vaccine as described above, as well as method of vaccination in pigs, and methods of treating and/or preventing associated diseases.
• Immunization includes the process of delivering an immunogen to a subject. Immunization may, for example, stimulate or induce or maintain a level of antibody and/or cellular response which can neutralize or kill or suppress a pathogen in the immunized animal, such as pig.
• NI-PoAstV-3 infections or associated diseases include various stages such as, in particular:
• Stage 1 Weaned Pig with Posterior Weakness: In milder case and/or in early stage of the disease, lesions are confined to the lumbar part of the spinal cord resulting posterior weakness with motor inco -ordination, pitching and fibrillary muscle twitching.
• Stage 2 Weaned Pig with Serious Tetraplegia: The cervical, dorsal and lumbar parts of spinal cord are equally affected producing serious movement disorder of both the front and hind legs. The consciousness is intact.
• Stage 3 Newly Weaned Pigs with End Stage Encephalomyelitis: The brainstem and cerebellum are most likely affected which result loss of consciousness, paresis, serious spastic paralysis of muscles. The animals react to pain, their sensory functions are retained.
• the invention may be used to suppress or reduce or slow or avoid any of these symptoms and/or to prevent progression from one stage to another, or to eliminate any pain and suffering, or to reduce viral load in an infected mammal.
• the method comprises administering a therapeutically effective dose of the vaccine.
• the vaccines of the invention can conveniently be administered intranasally, transdermally ( e., applied on or at the skin surface for systemic absorption), parenterally, ocularly, etc.
• the parenteral route of administration includes, but is not limited to, intramuscular, intravenous, intraperitoneal routes and the like.
• the dosage of the vaccine made according to the present invention depends on the species, breed, age, size, vaccination history, health status of the animal to be vaccinated, as well as of the route of administration, Le., subcutaneous, intradermal, oral intramuscular or intravenous administration.
• the vaccines of the invention can be administered as single doses or in repeated doses.
• the vaccines of the invention can be administered alone, or can be administered simultaneously or sequentially administered with one or more further compositions, such as for example other porcine immunogenic or vaccine compositions. Where the compositions are administered at different times the administrations may be separate from one another or overlapping in time.
• the vaccines or compositions containing the NI-PoAstV-3 immunogen are administered to a subject susceptible to or otherwise at risk for astro virus infection to enhance the subject own immune response capabilities.
• the subject to which the vaccine is administered is in one embodiment a pig (including adults or young piglets).
• the subject may be any animal (e.g., non human mammal) in which it may be desirable to elicit an immune response to the virus.
• Vaccines of the invention are preferably administered to pigs, adult pigs, but also to young pigs, piglets or to pregnant females. Vaccination of pregnant females is particularly advantageous as it confers passive immunity to the newborns via the transmission of maternal antibodies.
• the pigs may be less than 7, 6, 5, 4, 3, 2 or 1 week old; 1 to 6 weeks old; 2 to 5 weeks old; or 3 to 4 weeks old.
• the vaccine is administered to a subject who has not yet been exposed to the NI-PoAstV-3 virus.
• the subject is a pig which is in need of vaccination against NI-PoAstV-3- associated diseases.
• the present invention provides a container comprising an immunologically effective amount the vaccine as described above.
• the invention also provides vaccination kits comprising an optionally sterile container comprising an immunologically effective amount of the vaccine, means for administering the vaccine to pigs, and optionally an instruction manual including information for the administration of the immunologically effective amount the composition into pigs for treating and/or preventing NI-PoAstV-3 associated diseases.
• the invention provides a method of immunizing a pig against astroviral infection, or for preventing or treating an astrovirus-associated neurological disorder in a pig, comprising administering to the pig an immunogenically effective amount of a composition comprising any one or more of the following:
• the invention provides methods for immunizing or protecting a pig against at least one strain of porcine astrovirus, or for vaccinating a pig against a porcine astrovirus, comprising administering a vaccine or composition comprising an attenuated NI-PoAstV-3 strain.
• the invention provides methods for immunizing or protecting a pig against at least one strain of porcine astrovirus, or for vaccinating a pig against a porcine astrovirus, comprising administering a vaccine or composition comprising a NI- PoAstV-3 polypeptide, particularly an ORF2 protein or peptide.
• the invention provides methods for immunizing or protecting a pig against at least one strain of porcine astrovirus, or for vaccinating a pig against a porcine astrovirus, comprising administering a vaccine or composition comprising a vector comprising a nucleic acid encoding a NI-PoAstV-3 polypeptide, particularly an ORF2 protein or peptide.
• the methods of the invention result in an amelioration of one or more clinical symptoms of an infection, such as a reduction of viral load, a reduction of microscopic lesions, a reduction in the level of viral nucleic acid in tissues.
• the method also can result in a reduction or prevention of viral infection, as well as in an improvement in the condition of the treated animal, such as a reduction of neurological affection and improvement in mobility.
• the invention relates to a method of diagnosing the presence of an astrovirus (or of an anti-astrovirus antibody) in a mammal or in a sample, reagents used therefor, and diagnostic kits therefor.
• an astrovirus or of an anti-astrovirus antibody
• diagnostic kits therefor.
• nucleotide and amino acid sequences as described above, it is possible to produce reagents capable of recognizing porcine astro viruses of the invention in any sample.
• the skilled artisan can prepare primers or probes or epitopes or antibodies characteristic of such sequences, and which may be used to identify a NI-PoAstV-3 in a sample, e.g., by specific amplification, hybridization, ligand binding, or the like.
• an aliquot of a biological sample is contacted with a nucleic acid probe, a nucleic acid primer, or a ligand, characteristic of at least one NI-PoAstV- 3, and the presence or amount of complexes formed between said probe, primer, or ligand and analytes in the aliquot is determined.
• the probe or ligand may be in solution or immobilized on a support, such as a microarray.
• the biological sample may be treated prior to determination, e.g., diluted or concentrated or enriched for viruses or viral nucleic acid or anti- viral antibodies.
• detection is performed by selective hybridisation using nucleic probes, preferably immobilised on a support, such as a solid or semi-solid support having at least one surface, flat or not, for immobilising nucleic probes.
• a support such as a solid or semi-solid support having at least one surface, flat or not.
• Such supports are, for example, a slide, bead, membrane, filter, column, plate, etc. They can be made out of any compatible material, such as in particular glass, silica, plastic, fiber, metal, polymer, etc.
• the nucleic probes can be any nucleic acid (DNA, RNA, PNA, etc.), preferably single-stranded, with a sequence characteristic of (i.e., complementary and specific of a target region of) a viral RNA or DNA molecule.
• the probes typically comprise from 5 to 400 bases, preferably from 8 to 200, more preferentially less than 100, and even more preferentially less than 75, 60, 50, 40 or even 30 bases.
• the probes can be synthetic oligonucleotides, produced on the basis of the sequences of the target virus, according to standard synthesis techniques.
• the probe comprises all or part of a nucleic acid sequence selected from anyone of SEQ ID NOs: 1-4 or of a sequence complementary thereto.
• the nucleic acid probes has a length between 15 and 50 bases, more preferentially between 15 and 40 bases, and a sequence identical to a fragment of a sequence selected from anyone of SEQ ID NOs: 1-4 or the complementary strand thereof.
• the probe can also be designed in the opposite orientation.
• Such probes constitute another object of the present application, as well as the use thereof for diagnosing viral infection in a mammal.
• the method comprises contacting a sample to be analyzed to a support (such as a microarray) comprising at least one nucleic acid probe characteristic of a NI-PoAstV-3 nucleic acid under conditions allowing hybridization to occur, and detecting the presence of hybrids.
• a support such as a microarray
• the detection is performed by selective amplification using a primer or a pair of primers that amplifies all or part of a NI-PoAstV-3 nucleic acid.
• the primer can be specific for a target sequence within anyone of SEQ ID NOs: 1-4.
• the primer typically comprises a single-strand nucleic acid, with a length advantageously between 5 and 50 bases, preferably between 5 and 30 bases.
• Such a primer constitutes another object of the present application, as well as the use thereof (primarily in vitro) for diagnosing viral infection in a mammal.
• another object of the invention resides in the use of a set of nucleotide primers comprising at least 2 primers complementary to and specific of a target sequence in a nucleic acid selected from anyone of SEQ ID NOs: 1-4, or the complementary strand thereof, for in vitro or ex vivo diagnosis of viral infection in a mammal.
• Another particular embodiment comprises contacting a sample with a set of at least two nucleic acid primers, said at least two nucleic acid primers specifically amplifying a sequence characteristic of a NI-PoAstV-3 viral nucleic acid, and determining the presence of an amp licon.
• the method comprises contacting the sample with an anti-NI-PoAstV-3 virus antibody, and detecting the presence of immune complexes.
• the present invention further relates to a method of generating an antibody which is capable of binding to a NI-PoAstV-3 virus or sub-units thereof.
• the method may comprise immunizing an animal, such as a rabbit, guinea pig, or rodent and harvesting the antibody produced thereby.
• the antibodies of the invention may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, or may be derivatives thereof such as hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies (Fab')2 fragments, F(ab) fragments, Fc fragments, single- domain antibodies, dimeric or trimeric antibody fragments or constructs, or functional fragments thereof which bind to the antigen in question.
• Antibodies may be produced using techniques well known to those of skill in the art and disclosed in "A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, N.Y. (1988)".
• a particular embodiment of the invention is an antibody, or a derivative thereof having essentially the same antigen specificity, which binds an ORF2 polypeptide as defined in the present invention.
• Diagnostic kit thus may comprise nucleic acid probes or primers, antigens and/or antibodies specific for a NI-PoAstV-3 virus, and diagnostic testing may be performed on a sample of physiological fluid (blood, plasma, serum and the like) or a sample of tissue (ganglia, liver, lungs, kidneys and the like) obtained from a pig to be tested.
• tissue samples were collected from 6 paraplegic pigs and five asymptomatic animals (Table 1, 2), nasal and anal swab pairs from another 5 paraplegic and 13 healthy animals were also tested.
• the tissue samples were washed twice in 0.1 M phosphate buffered saline (PBS) to remove the excess blood and held in -80oC until total RNA extraction.
• the nasal swabs were acquired using two polyester-tipped swabs.
• the dissected samples (Table 2) were fixed freshly with buffered 8% of formaldehyde, dehydrated and embedded into paraffin.
• the three swine farms are located in the central and eastern parts of Hungary, approximately 100 km distance from each other without known connection.
• the astrovirus sequences were aligned using MUSCLE web tool of EMBL-EBI.
• the pairwise nucleotide (nt) and amino acid (aa) identity calculations of the aligned sequences were performed using GeneDoc ver.2.7.
• Phylogenetic trees of deduced amino acid sequence alignments were constructed using MEGA software ver. 6.06.
• the Neighbor- Joining method with the Jones-Taylor-Thornton matrix-based model was used for the construction of the trees. Bootstrap values were set up to 1000 replicates and only likelihood percentages of more than 50% were indicated.
• BS Brainstem
• SC spinal cord
• NS nasal swab
• faecal samples were collected from a newly weaned pig (GD-1, index animal) showing signs of encephalomyelitis and posterior paraplegia (Stage 1) from index GD farm.
• the BS and SC samples tested negative by RT-PCR for the presence of pestivirus (family Flaviviridae), and several swine-infecting picornaviruses (family Picornaviridae). 71ie presence of astro virus was investigated using pan-astro virus (Pan-AstV) PCR primers (31).
• Pan-AstV pan-astro virus
• Pan-AstV PCR products were sequenced and compared to each other and to the available astroviruses using BLASTn.
• the 397-nt-long sequences of BS and SC were identical and showed 89%> nt identity to a porcine astrovirus 3 strain US-M0123 (JX556691) as the closest match (32).
• the full-length genome of the neuroinvasive astrovirus strain NI-Brain/9-2016a/HUN (KY073229) from the BS sample and the complete capsid-encoding ORF2 from the SC sample NI-SC/9-2016a/HUN (KY073230) both from the index animal were sequenced (see SEQ ID NO: 1 and SEQ ID NO: 2).
• the 6393-nt-long (without the poly(A)-tail) complete genome showed the typical astrovirus genome organization with three, 2529 nt, 1527 nt and 2265 nt long putative open reading frames (ORFla, ORFlb and ORF2) flanked by short 5' and 3' untranslated regions (Fig. 1).
• the conserved proteolytic cleavage site (V561HQJ,TNT) of serine protease (ORFla) and the conserved Y358GDD motif of the RdRp (ORFlb) were identified (33).
• ORFla 842 aa
• ORFlb 508 aa
• capsid protein of ORF2 754 aa
• the 3'UTR of NI-Brain/9-2016a/HUN is 27 nt shorter and does not contain the short sequence repeat found at the 3' end of strain US-MO 123 (G6381 /6392 AUUUCUUUNA) .
• NI- Brain/9-2016a/HUN strain most likely belongs to the PoAstV-3 genotype.
• the ORF2 of NI-Brain/9-2016a/HUN shares 99% nt/aa identity to the corresponding capsid gene of NI-SC/9-2016a/HUN from the spinal cord of the same animal suggests that the same virus was present in both regions of the CNS.
• PoAstV-3 was also detectable using RT-PCR in all CNS samples collected from another four affected newly weaned pigs held in the index farm (Table 1). All of the samples from the asymptomatic control animals were NI-PoAstV-3 negative. The complete genomes of two additional PoAstV-3 strains (NI-Brain/173-2016a/HUN, KY073231 and NI-Brain/386-2015/HUN, KY073232) originated from another two affected animals (GD-3 and GD-5) in Stage 3 of the disease were also determined (SEQ ID NO: 3 and 4).
• PoAstV-3 was detectable in multiple non-CNS samples from the respiratory system, the lymphoid system, the circular system and salivary glands of affected animals (Table 1). PoAstV-3 was detected only in a single ileum sample and two of the three analyzed faecal samples using RT- nested (n) PCR (Table 1). Samples from internal organs such as spleen, and kidney as well as the urine samples tested negative by RT-nPCR (Table 1). The copy number of PoAstV-3 was determined using SYBR Green-based RT-qPCR. All of the PoAstV-3 RT-nPCR positive samples were negative by qPCR indicating low copy number ( ⁇ ca.
• the nasal and anal swabs of the asymptomatic animals were all RT-nPCR negative. Due to the variable amounts of samples collectable by polyester-tipped swabs which were unable to measure accurately the absolute quantification of PoAstV-3 by RT-qPCR of nasal and anal swabs were not performed.
• the capsid genes primers are: first round: PoAstV3-CAP-R-out 5'- GGC CCA GTR TTA GGB GCA TT-3' (SEQ ID NO: 22) /PoAstV3-CAP-F-out 5'- ACC ATY TGG CAG ATA GTK GA-3' (SEQ ID NO: 23); second/nested round: PoAstV3-CAP-R-in 5'- CCA GTR TTA GGB GCA TTA ATC TG-3' (SEQ ID NO: 24) / PoAstV3-CAP-F- in 5'- CAT YTG GCA GAT AGT KGA TGA-3 ' (SEQ ID NO:25).
• the spinal cord FFPE sample from Balmazujvaros was negative using both RT-nPCR primer sets.
• the RT-nPCR positive samples were also positive by in situ hybridization (ISH) (data not shown).
• Hybridization was distinct, with punctate to diffuse cytoplasmic staining throughout the cytoplasm.
• the unique microarchitecture of the Purkinje layer of the cerebellum offered the clear demonstration that viral nucleic acid was present within dendritic processes coursing through the molecular layer (Fig. 4G,J). There were no pathological lesions found in other samples from kidneys, liver, gastrointestinal tract or immune system. The samples from the latter were also negative by PoAstV-3 ISH (Table 2). Discussion
• Astrovirus RNA was detected in multiple tissues from newly weaned pigs in Hungary with encephalomyelitis and posterior paraplegia of unknown origin with the highest viral load detected in brain and spinal cord. The same virus was also detected in brain and spinal cord FFPE samples from similarly affected animals from two additional swine herds collected between 2011 and 2014. These data define a genetically similar, neurovirulent astrovirus circulating in multiple swine farms in Hungary.
• the Ni-PoAstV-3 and the observed encephalitis and paraplegia are in a probable causal relationship (Level 2).
• neurological signs were observable mainly among newly weaned pigs.
• the time of weaning which include nutritional- (from milk to solid feed), social-, (mixing with different litters without the sow) and environmental changes (moving to a new pen) is known to be the most stressful period in the lifetime of pigs, and is associated with dysfunction of the immune system.
• the identified astrovirus strains belong to the PoAstV-3 genotype which clusters within the VA/HMO phylogenetic clade.
• PoAstV-3 strains were detected only from faecal samples of healthy or diarrhoeic piglets, worldwide.
• the PoAstV-3 was either undetectable or present only at low viral loads in the analyzed faecal samples while the virus was generally detectable in the respiratory system of paraplegic pigs. This may indicate that CNS infection/replication occur at a later date than enteric replication or that initial replication occurs extra-intestinal, e.g. in the respiratory tract.
• PoAstV-3 The highest viral loads of PoAstV-3 were measured in brain and spinal cord samples similar as found in diseased ovine as well as human patients with astrovirus-associated encephalitis. PoAstV-3 was also detectable in the sera and multiple organs of the respiratory-, lymphoid- and cardiovascular systems of diseased swine. These results indicate that PoAstV-3 can result in viremia and disseminated infection involving the brain, spinal cord and multiple organs during the acute phase of encephalomyelitis and posterior paraplegia. Astrovirus there seem to plays a role in a common and severe disease (encephalomyelitis and paralysis) in pigs.
• Example 3 Virus inactivation using chemical agent
• EI inactivating agent ethylenimine
• Inactivation of the NI-poAst-3 virus suspension is done with the concentration of 0.05% (w/v) and 0.075% (w/v) at the temperature of 23°C and 37°C for 24 hours and 48 hours.
• the pH value of the EI stock solution is adjusted to 8.0-8.5 before being used for inactivation.
• Inactivation is stopped by adding 50%> (w/v) sodium-thiosulphate solution to the virus suspension in 1.5M excess then on the following day the pH is adjusted by adding glycin-HCl buffer to 7.2-7.4.
• the NI-PoAstV-3 virus can be fully inactivated according to the appropriate inactivation control method irrespectively of the concentration, temperature and time applied during the inactivation.
• Example 4 Preparation of a composition with adjuvant (10% oil)
• a NI-PoAstV-3 virus vaccine is prepared as follow: First Step: A high shear rotor stator emulsifier is used to produce the formulations. To produce an emulsion, one volume of oily phase is emulsified at 25°C with one volume of aqueous phase #1. The aqueous phase is added to the oily phase under agitation, 5000 rpm (rotation per minute) for 1 minute. The rotation speed is progressively increased with the augmentation of the volume to 10000 rpm during 1 minute. During this step the emulsion is a water-in-oil emulsion. For the emulsion, phase composition is as follows:
• Aqueous phase #1 (200 ml):
• Sorbitan monooleate and sorbitan trioleate (20 OE) are introduced in the oily phase.
• a 20% (w/v) solution of sorbitan monooleate (20 OE) is prepared in the same buffer as the vaccine, for example, in phosphate disodic and monopotassic 0.02M isotonic buffer.
• the emulsion changes to an oil-in-water emulsion.
• the emulsion is then placed in a cold chamber at 5°C for at least 4 hours. At this stage, the emulsion is a pre-emulsion containing 50% of oily phase.
• the aqueous phase #2 is prepared with 200 ml of phosphate disodic and monopotassic 0.02M isotonic buffer pH 7.2-7.6 with NI-PoAstV-3.
• the pre-emulsion as prepared in the first step is cooled to about 5°C, diluted by adding half the volume of the aqueous phase #2 at the same temperature, and mixed by the rotation of a magnetic bar for 1 minute.
• Piglets are vaccinated with the NI-PoAstV-3 vaccine with the adjuvant as prepared in Example 5 (Group A) on day 0 (7-week-old pigs).
• a first control group receives the adjuvant as described in Example 5, only (Group B).
• a second control group receives PBS only (Group C).
• a 2 ml volume is injected by intramuscular route.
• a second injection of vaccine or PBS is performed on day 21 (10-week-old pigs).
• 7 pigs from each group are challenged by intranasal administration of 2 ml NI-PoAstV-3 virus suspension containing 10 3 TCID50 of NI-PoAstV-3 virus/ml.
• piglets are weighed on days 55 and 72. Rectal temperatures are recorded. Blood samples are taken 7 days before the first vaccination (day -7), at the time of the second vaccination (day 21), at challenge (day 42) and on days 55, 64 and 72 for immunofluorescence serology. Sera are tested individually, diluted twofold, starting with 1 :2 in PBS supplemented with 0.5%> bovine serum albumin. LIST OF SEQUENCES
• SEQ ID NO: 12 KY073229 ORFlab (amino acid)
• SEQ ID NO: 4 KY073230 ORF2 (nucleotide)
• SEQ ID NO: 10 KY073231 ORFla (amino acid)
• SEQ ID NO: 7 KY073231 ORF2 (amino acid)
• SEQ ID NO: 2 KY073231 Genome (nucleic acid)
• SEQ ID NO: 14 KY073232 ORFlab (amino acid)
• SEQ ID NO: 8 KY073232 ORF2 (amino acid)
• SEQ ID NO: 3 KY073232 Genome (nucleic acid)
• Table 1 Results of the porcine astro virus 3 RT-nested (n)PCR screening of different tissues collected from 5 affected pigs with encephalomyelitis and posterior paraplegia (PP) from the index farm. + or - signs indicate the results of the first-; (+) or (-) signs indicate second (nested) reactions.
• the screening RT-(n)PCR primers are designed to the RdRp region of porcine astrovirus 3 (Fig.l). n.a.: No 494 available sample. LN: lymph node.*: Nasal swab samples
• Serum n.a. n.a. + + n.a. Table 2 List of FFPE samples used for porcine astrovirus 3 (PoAstV-3) detection, histology and in situ hybridization (ISH) analyses.
• GD index farm.
• RdRp RNA-dependent RNA polymerase
• RdRp and capsid RT-nested (n) PCR + or - signs indicate the results of the first-; (+) or (-) signs indicate second (nested) round of RT-PCR screening reactions.
• the screening RT-PCR primers are designed to either the RdRp or the capsid region of porcine astrovirus 3 (Fig.l).
• the results of RT-(n)PCR screening reactions refer to a mixture of tissues embedded into the total of seven paraffin blocks.
• ISH + PoAstV-3 ISH positive tissue samples
• ISH - PoAstV-3 ISH negative samples.
• Table 3 Amino acid (aa) differences of the "neuro invasive" (“Ni") study strains of porcine astrovirus 3 (PoAstV-3): NI-Brain/9-2016a/HUN (KY073230), NI-Brain/173-2016a/HUN (KY073231) and NI-Brain/386-2015/HUN (KY073232) compared to the available PoAstV- 3 strains detectable only from faecal samples (referred as "enteric” or "Ent” strains): JX556691, LC201595-7, LC201599.
• AD Presumed particle assembly domain
• RID Presumed Receptor- Interaction domain.

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