Identification of Human Sarcoma Virus

Abstract

The present invention relates to the discovery of the first pathogenic human sarcovirus in samples from human hepatitis patients, the capsid of which shows only 39% identity at the amino acid level to the most closely related known sarcovirus. This discovery enables the generation of recombinant nucleic acids, primers, peptides, probes, antibodies, immunogenic compositions specific for human sarcovirus and the generation of diagnostic assays.

Description

Technical Field

[0001] The present invention relates to the detection or diagnosis of human circovirus infection, including determining the presence in a biological sample of at least one nucleic acid or protein of said virus or an antibody thereto. The present invention also relates to various diagnostic agents derived from viral nucleic acids or proteins, in particular nucleic acid primers and probes, antigens and antibodies, and their use for the diagnosis of viral infections and related diseases caused by circovirus, in particular hepatitis. The present invention further relates to an antigen derived from a viral protein as a vaccine for the prevention of circovirus infections and related diseases, in particular hepatitis.

Background Art

[0002] Circovirus is a small virus in the family Circoviridae having a circular single-stranded DNA genome of about 2 kb. Hui et al., Viruses 2021, 13, 944. Circovirus is associated with a variety of diseases ranging from asymptomatic to lethal in birds, mammals, reptiles, and fish. Id. Pathogenic human circovirus has not been described to date.

Summary of the Invention

[0003] Human hepatitis is a potentially lethal disease that is a health problem affecting millions of people worldwide with high mortality rates. Hepatitis A virus (HAV) causes acute self-limiting hepatitis, while all liver-directed viruses (hepatitis B, C, D, and E viruses (HBV, HCV, HDV, and HEV)) can cause chronic infections. According to the CDC, acute hepatitis not diagnosed as being caused by a known virus "may be caused by exposure to certain medications, drugs, alcohol, and toxins. It may also be caused by autoimmune diseases. Other possible causes of non-viral hepatitis include contaminated water or food, nutritional supplements and herbal supplements, traditional or home remedies, wild mushrooms and plants, and chemicals such as metals, solvents, paint thinners, or pesticides. Investigation of Acute Non-Viral Hepatitis of Unknown Etiology Potentially Associated with an Alkaline Water Product, CDC, 2021. In addition, there may still be unknown viruses involved. Therefore, the validity of diagnosis is essential for the proper management of the disease. In this context, there is a need in the art to identify new pathogens causing hepatitis that can improve the range of possible diagnostic tests and enhance the efficiency of diagnosis. The present invention meets this need in the art.

[0004] The present invention will be more fully understood by reference to the drawings.

Brief Description of the Drawings

[0005]

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[0006] The novel human circovirus was identified in samples from human hepatitis patients. The capsid of the novel human circovirus shows only 39% identity at the amino acid level to the most closely related circovirus, Wolvfec circovirus, Genbank QSX73454. Phylogenetic analysis of the capsid gene indicates that this virus corresponds to a new clade within the members of the genus Circovirus.

[0007] The convergence of virological and clinical data indicates that the inventors have identified in France a new liver-directed virus belonging to a group of viruses that until now have only been represented by animal pathogens. HCirV-1 can cause less severe, unclassified forms of hepatitis or other diseases in humans or even other vertebrates.

[0008] The unique properties of the HCirV-1 DNA and protein sequences enable the production of recombinant nucleic acids, expression vectors, primers, peptides, probes, antibodies, immunogenic compositions specific for human circoviruses that do not amplify or detect other circovirus genus members, as well as the creation of kits and diagnostic assays.

[0009] Recombinant nucleic acid The present invention encompasses recombinant nucleic acids comprising HCirV-1 nucleic acid sequences. In one embodiment, the recombinant nucleic acid comprises an HCirV-1 capsid or replicase sequence.

[0010] In one embodiment, the recombinant nucleic acid of the present invention comprises or consists of the following nucleic acid sequence corresponding to the entire genome of HCirV-1, which encodes the capsid protein and replicase protein of HCirV-1:

[0011]

[0012] This array is currently registered under accession number ON677309.

[0013] In one embodiment, the invention encompasses variants thereof having 65%, 70%, 80%, 90%, 93%, 95%, 97%, 98%, more than 99%, or 100% identity to SEQ ID NO: 10.

[0014] Certain variants are, for example, the hCirV-2 viruses recently registered under accession numbers ON226770 and OP744467 (Li et al., Emerging Infectious Diseases, Vol. 29, issued on May 5, 2023). These variants share 68% and 79% identity with SEQ ID NO: 10, respectively.

[0015] The recombinant nucleic acid of the invention can comprise all or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides identical to the nucleotide sequence of SEQ ID NO: 10.

[0016] In certain embodiments, the recombinant nucleic acid of the invention comprises or consists of the following nucleic acid sequence corresponding to the sequence of HCirV-1 encoding the capsid protein of HCirV-1:

[0017] ATGGCTAGATATACCCTCAGGAGGAGAAGACCCCGCCCCAGGAGGCGTGCCCGCAGATTTCGACGAATCAGGAGACGCCGCATTTACATAAGACGTCCCACCAATAATGGAACAGTTGTCCAAAAATTTTGCAAGACTGAACCCTTCACCCTCCAAAACCAAGATTCAACCAATGCCAAAAAATGGATGCAAGGCTCTTACACAACCACCCTCAGTGAATGGGTTTCCTCACCAGGCTGGGACTTTTATAAAATCCTAAAACTCAAACTAACCTTCCTTCCCTTCAATGCCACACAGCGAATGAACTACTGGCCTATAGGAAACACCATCATAGACCTGGATGGACCCTGGAATGAACCTAACACAGTACAATCAAACCTACTCTGCAACACTGCCACTTCCAGACTTTGGAAGGCTAACAGAAAGCATAGCAGATATTTTACTCCTCGCCCTCTCATCCATCTTACTGGCAATACTACTTCTTCTCAAGCATCAGCTCTCCTCCCACGCAGAACCTGGTGGAACTCTGCAGAATCTACTGTCAAATGGGGAGGACTCAAGTACTCTCATTACGTTGAATATAATCAACCAGTCAGATACTGGCTCATCAAATCAGCCTGGATCAAATGGAAATACCTTCTCTAA(SEQ ID NO: 1)

[0018] In one embodiment, the present invention includes variants thereof having 65%, 70%, 80%, 90%, 93%, 95%, 97%, 98%, more than 99%, or 100% identity to SEQ ID NO: 1.

[0019] In some embodiments, the recombinant nucleic acid of the present invention comprises a fragment of SEQ ID NO: 1 that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of SEQ ID NO: 1.

[0020] In another embodiment, the recombinant nucleic acid comprises or consists of the following nucleic acid sequence encoding the replicase protein of HCirV-1:

[0021] ATGCAGCGGGAGAGCCCAGTGGCGAGGTGGTGTTTTACGATAAACAACTGGACCCAAGAAGAGTTGGATTGCTTGCTGCAGTGTGGCAAAGAGAAAGCGAAGTATCTTTGTATCGGCAGAGAGGTTGGAAGCGGCGGTACCCGGCACTTGCAAGGATACGTGAATTTCAAGAACAAGCGAAGAATGAGACAGGTAAAGGCCTTGCCTGGTTTTATGCGTGCTCACTTAGAAGCGGCCAAGGGGACCGAGAAGCAGGCGAGCGACTACTGCAAGAAAGACGGCGATTACCTGGAGATTGGAGAGAGTGGCTGTGCGGGGCGCCGACTGGATTTGGAGACAGCAGCGAAGATACTGACTGAGTCTGGCGGGAACTTGAAGTGTGTTGCCGAGGCTTGTCCCGGGGTTTATATAAAGTATGGGCGGGGTTTGAGGGATTACGCAAGTGTTATGAAATTAAAGCAACCTCGCAATTTTAAGAGTACTGTGATTGTTATATGTGGGGATCCTGGTTGTGGGAAAACTAGATATGTTATGGAGGATTGTAGGGAGAAAGGGTTAAGCATGTATTGGAAGCCAAGGGGAATTTGGTGGGATGGCTATGATGGGGAGGATGTGGTGGTGTATGATGATTTTTATGGGTGGGTTCCTTATGATGAGATGTTGAGGGTTATGGATAGGTATCCTTTGAAGGTTCCTGTTAAGGGGGGTTATGTGGATTTTACATCTAAGATTATATATGTGACAAGTAATGTGAAGCCTGAGGATTGGTATAGTAGTGAGAATATTAAGGGGAAGTTGGAGAGTTTGTTTAGGCGGATCAATGTTTATCTGGAGGGTTTTGGCGGGGAGATCCGTGTGGGAACGCCTCTGTATCCTATTAACTATTAA (SEQ ID NO: 11)

[0022] In one embodiment, the invention encompasses variants thereof having 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98%, more than 99%, or 100% identity to SEQ ID NO: 11.

[0023] The recombinant nucleic acid of the invention can comprise all or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 consecutive nucleotides identical to the nucleotide sequence of SEQ ID NO: 11.

[0024] In one embodiment, the recombinant nucleic acid of the invention comprises an origin of replication for replication in bacteria or yeast.

[0025] In one embodiment, the recombinant nucleic acid of the invention comprises a heterologous sequence that enables expression, such as a heterologous promoter or enhancer.

[0026] In one embodiment, the recombinant nucleic acid of the invention encodes the following amino acid sequence or a fragment thereof of the capsid protein of HCirV-1:

[0027] MARYTLRRRRPRPRRRARRFRRIRRRRIYIRRPTNNGTVVQKFCKTEPFTLQNQDSTN AKKWMQGSYTTTLSEWVSSPGWDFYKILKLKLTFLPFNATQRMNYWPIGNTIIDLDGP WNEPNTVQSNLLCNTATSRLWKANRKHSRYFTPRPLIHLTGNTTSSQASALLPRRTW WNSAESTVKWGGLKYSHYVEYNQPVRYWLIKSAWIKWKYLL (SEQ ID NO: 2)

[0028] In one embodiment, the recombinant nucleic acid of the present invention encodes a protein having at least 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2.

[0029] In another embodiment, the recombinant nucleic acid of the present invention encodes the following amino acid sequence of the replicase protein of HCirV-1 or a fragment thereof:

[0030] MQRESPVARWCFTINNWTQEELDCLLQCGKEKAKYLCIGREVGSGGTRHLQGYVNFKNKRRMRQVKALPGFMRAHLEAAKGTEKQASDYCKKDGDYLEIGESGCAGRRLDLETAAKILTESGGNLKCVAEACPGVYIKYGRGLRDYASVMKLKQPRNFKSTVIVICGDPGCGKTRYVMEDCREKGLSMYWKPRGIWWDGYDGEDVVVYDDFYGWVPYDEMLRVMDRYPLKVPVKGGYVDFTSKIIYVTSNVKPEDWYSSENIKGKLESLFRRINVYLEGFGGEIRVGTPLYPINY (SEQ ID NO: 12)

[0031] In one embodiment, the recombinant nucleic acid of the present invention encodes a protein having at least 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identity to either the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 12.

[0032] Expression vector The present invention encompasses recombinant vectors for the expression of HCirV-1 protein. The recombinant vector can be a vector for eukaryotic or prokaryotic expression, such as a plasmid, a phage for bacterial introduction, a YAC capable of transforming yeast, a viral vector, especially a retroviral vector, or any expression vector. The expression vector defined herein is selected to enable the production of HCirV-1 protein or polyepitope either in vitro or in vivo.

[0033] In one embodiment, the expression vector of the present invention encodes a protein having at least 50%, 60%, 70%, 80%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2 or SEQ ID NO: 12.

[0034] In one embodiment, the expression vector of the present invention encodes a protein having 50%, 60%, 70%, 80%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identity to any of the sequences in SEQ ID NO: 2 or SEQ ID NO: 12.

[0035] In one embodiment, the expression vector of the present invention encodes a protein purification tag. In one embodiment, the expression vector encodes a protease cleavage site, such as a TEV cleavage site, inserted between the HCirV-1 protein coding sequence and a protein purification tag such as a polyHis tag. In a preferred embodiment, the expression vector encodes a His tag. In one embodiment, the protease cleavage site is arranged to remove the His tag, for example, after purification.

[0036] The expression vector of the present invention can include a transcription control region (including a promoter, an enhancer, a ribosome binding site (RBS), a polyA signal), a termination signal, a prokaryotic or eukaryotic origin of replication, and / or a selectable gene. The characteristics of the promoter, i.e., constitutive, transient or inducible (e.g., IPTG), strong or weak, tissue-specific and / or development stage-specific promoter, can be easily determined by those skilled in the art considering the required expression. The vector can also include a sequence that enables conditional expression, for example, a sequence of the Cre / Lox system or a similar system.

[0037] In various embodiments, the expression vector of the present invention is a plasmid, a phage for bacterial introduction, a YAC capable of transforming yeast, a viral vector, or any expression vector. The expression vector defined herein is selected to enable the production of a protein or polyepitope either in vitro or in vivo.

[0038] The nucleic acid molecule or vector according to the present invention can be obtained by conventional methods known per se according to standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc., Library of Congress, USA). For example, they may be obtained by amplification of the nuclear sequence by PCR or RT-PCR, or alternatively by total or partial chemical synthesis.

[0039] The vector of the present invention is constructed by conventional recombinant DNA and gene manipulation methods known per se and introduced into a host cell. A number of vectors into which a nucleic acid molecule of interest can be inserted for introducing and maintaining the nucleic acid molecule of interest into a host cell are known per se, and the selection of an appropriate vector depends on the intended use for this vector (e.g., replication of the sequence of interest, expression of this sequence, maintenance in chromosomal form of the sequence or alternatively integration into the chromosomal material of the host), and the nature of the host cell.

[0040] The present invention further encompasses a cell comprising the vector of the present invention.

[0041] The present invention also encompasses a method for preparing a protein, which includes culturing a cell comprising the expression vector of the present invention and recovering the expressed protein.

[0042] The present invention further encompasses a protein produced from the nucleic acid of the present invention by these methods.

[0043] Primers and probes The present invention encompasses primers and probes based on the HCirV-1 nucleic acid sequence of the present invention described above. In one embodiment, the primers and probes of the present invention are based on the HCirV-1 capsid or replicase sequence.

[0044] In one embodiment, the primers and probes of the present invention comprise or consist of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, or 150 consecutive nucleotides identical to the nucleotide sequence of SEQ ID NO: 1.

[0045] Primers preferred for the amplification of HCirV-1 nucleic acid are HCirV1-Fw1: ACCTGGATGGACCCTGGAAT (SEQ ID NO: 4), HCirV1-Rv1: AGAGTTCCACCAGGTTCTGC (SEQ ID NO: 5), HCirV1-Fw2: TTCGACGAATCAGGAGACGC (SEQ ID NO: 6), HCirV1-Rv2: GGCAGTGTTGCAGAGTAGGT (SEQ ID NO: 7), HCirV1-Fw3: CAGGAGACGCCGCATTTACA (SEQ ID NO: 8), and HCirV1-Rv3: CCCATTCACTGAGGGTGGTT (SEQ ID NO: 9) are as follows.

[0046] These three sets of PCR primer pairs (SEQ ID NOs: 4-5, SEQ ID NOs: 6-7, and SEQ ID NOs: 8-9) were designed to amplify short (less than 500 nt) fragments in the capsid gene (i.e., the six individual primers corresponded to three forward primers and three reverse primers). Considering that circoviruses are small circular DNA viruses, these primers can also be used in various combinations for inverse PCR experiments to amplify long (more than 500 nt) fragments. Alternatively, displacement amplification enzymes such as Phi29 polymerase can also be used for genome amplification, detection, and genome finishing.

[0047] Other TaqMan primers and probes preferred for amplification or labeling of HCirV-1 are as follows: HCirV-Fw: 5’-TTAGAAGCGGCCAAGGGGA-3’ (SEQ ID NO: 13) HCirV-Rv: 5’-CGCTGCTGTCTCCAAATCCA-3’ (SEQ ID NO: 14) HCirV-Pb: 5’-[FAM]GGAGATTGGAGAGAGTGGCTG[BHQ1]-3’ ([FAM]-SEQ ID NO: 15-[BHQ])

[0048] The sequences of these primers and probes are designed to specifically detect the replicase protein of human circovirus without detecting other non-human circoviruses. The resulting amplicon has a size of approximately 120 base pairs.

[0049] Preferably, the primer or probe of the present invention is labeled with a fluorescent label, a radioactive label, or an enzyme label.

[0050] The primer and / or probe of the present invention can comprise or consist of all or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, or 150 consecutive nucleotides identical to any of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 10, or SEQ ID NO: 11.

[0051] The primer and / or probe of the present invention can comprise or consist of all or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of SEQ ID NOs: 4-9 or SEQ ID NOs: 13-15.

[0052] The present invention encompasses the primers and probes of the present invention for use in diagnostic assays and their use in such assays.

[0053] Nucleic acid detection and amplification kit The present invention encompasses nucleic acid detection and amplification kits containing the probes and / or primers of the present invention, such as those disclosed above.

[0054] Preferably, the primer or probe of the present invention is labeled with a fluorescent, radioactive, or enzyme label.

[0055] Preferably, the kit of the present invention contains amplification and / or hybridization reagents. For example, the kit of the present invention can contain dNTPs (dATP, dCTP, dGTP, and dTTP), buffer, and reverse transcriptase and / or heat-stable polymerase (such as Taq polymerase).

[0056] Nucleic acid diagnostic assay The present invention encompasses nucleic acid diagnostic assays that utilize the probes and / or primers of the present invention.

[0057] The present invention encompasses methods for the specific detection of HCirV-1.

[0058] In one embodiment, the method of the present invention includes preparing a sample, contacting the sample with a probe of the present invention, and detecting the presence or absence of HCirV-1 nucleic acid in the sample by conventional techniques in the art.

[0059] In one embodiment, the sample can be subjected to an amplification reaction that increases the amount of HCirV-1 nucleic acid in the sample for detection.

[0060] In one embodiment, the method of the present invention includes preparing a sample, optionally subjecting the sample to a reverse transcription reaction to generate a cDNA copy of HCirV-1 RNA in the sample using a "reverse primer" specific for HCirV-1 RNA, amplifying the HCirV-1 DNA using a "reverse primer" and a "forward primer", and detecting any amplified product. In one embodiment, the amplified product is detected using a probe. The method can be used for determining whether HCirV-1 is present in the sample.

[0061] A preferred primer set for the amplification of HCirV-1 nucleic acid is HCirV1-Fw1 (SEQ ID NO: 4) and HCirV1-Rv1 (SEQ ID NO: 5), HCirV1-Fw2 (SEQ ID NO: 6) and HCirV1-Rv2 (SEQ ID NO: 7), and HCirV1-Fw3 (SEQ ID NO: 8) and HCirV1-Rv3 (SEQ ID NO: 9) are.

[0062] Other preferred primer sets are HCirV-Fw: 5’-TTAGAAGCGGCCAAGGGGA-3’ (SEQ ID NO: 13) HCirV-Rv: 5’-CGCTGCTGTCTCCAAATCCA-3’ (SEQ ID NO: 14) are.

[0063] In a preferred embodiment of the assay of the present invention, the sample is a biological sample, e.g., feces, saliva, blood, plasma, serum, urine, cerebrospinal fluid, or tissue, preferably a liver sample. In some embodiments, the sample is a human or animal clinical sample (i.e., a sample from a living or dead individual (human or animal) suspected of having a circovirus virus). In another embodiment, the sample can be an environmental sample (sewage).

[0064] The sample can be subjected to well-known isolation and purification protocols or can be used directly. For example, the sample can be subjected to treatments for releasing / extracting the nucleic acid of the sample and / or for removing the protein and other non-nucleic acid components of the sample using conventional techniques.

[0065] Amplification of HCirV-1 genomic DNA can be carried out using two primers that are both specific for HCirV-1, namely, a “forward” and a “reverse” primer.

[0066] Reverse transcription of the RNA of HCirV-1 can be carried out using a “reverse primer” specific to HCirV-1. A “reverse primer” is a primer that, based on its 5’-3’ orientation, can bind to single-stranded RNA and play a role in initiating the generation of a complementary DNA (cDNA) copy of that RNA. Reverse transcription can be achieved using well-known and conventional methods. The reaction mix for reverse transcription contains reagents for the reaction, such as a reverse primer, dNTPs (dATP, dCTP, dGTP, and dTTP), a buffer, and a reverse transcriptase. Exemplary reaction conditions are described in the Examples.

[0067] Amplification of the cDNA copy of HCirV-1 generated by reverse transcription can be carried out using a “forward primer” specific to HCirV-1. A “forward primer” is a primer that, based on its 5’-3’ orientation, can bind to the single-stranded antisense cDNA copy of the RNA generated by reverse transcription and play a role in initiating the generation of a double-stranded DNA copy of that RNA. Amplification can be achieved using well-known and conventional methods. The reagent mix for amplification contains reagents for the reaction, such as a forward primer, a reverse primer, dNTPs, a buffer, and a DNA polymerase.

[0068] In one embodiment, the method of the present invention is carried out using a single RT-PCR reagent mix containing reagents for the reverse transcription reaction and the amplification reaction. Preferably, the reverse primer used for the reverse transcription reaction is also used for the amplification reaction.

[0069] Preferably, the reverse transcription and amplification reactions are carried out in a plastic or glass container, most preferably the same container.

[0070] Known amplification methods in the art include RCA, MDA, NASBA, TMA, SDA, LCR, b-DNA, PCR (including all types of RT-PCR), RAM, LAMP, ICAN, SPIA, QB replicase, or Invader. A preferred amplification method is polymerase chain reaction (PCR) amplification. See, for example, PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (ed. Iinis et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (ed. McPherson et al., IRL Press, Oxford); and U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,333,675. A more preferred PCR method is real-time PCR, PCR-HRM (high-resolution DNA melting) (see Andriantsoanirina et al., Journal of Microbiological Methods, 78:165 (2009)), and PCR combined with a ligase detection reaction based on fluorescent microspheres (Luminex® microspheres).

[0071] As amplification techniques, in particular, isothermal methods and PCR-based techniques can be mentioned. As isothermal techniques, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA), exponential amplification reaction (EXPAR), isothermal chimeric primer-initiated nucleic acid amplification (ICAN), signal-mediated RNA amplification technology (SMART), and the like can be mentioned (for example, see Asiello and Baeumner, Lab Chip; 11(8): 1420-1430, 2011).

[0072] Preferably, the PCR technique quantitatively measures the starting amount of DNA, cDNA, or RNA. Examples of PCR-based techniques according to the present invention include, but are not limited to, techniques such as quantitative PCR (Q-PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative reverse transcriptase PCR (QRT-PCR), or digital PCR. These techniques are well known to those skilled in the art and are readily available techniques. Preferably, Q-PCR is carried out using the primers described in the examples, preferably the primers described in the examples of this specification.

[0073] Preferably, the method of the present invention is a one-step real-time RT-PCR assay. Preferably, a probe is used to detect the amplified product. The probe can be labeled with fluorescence, radioactivity, or an enzyme label. The amplified product can be detected using a specific detection chemistry, for example, fluorescence resonance energy transfer (FRET) probes, TAQMAN probes, molecular beacons, scorpion probes, fluorescently labeled (or other labeled) primers, light-up probes, or dyes-based chemistries, DNA, PNA, LNA, or RNA containing modified bases that bind to the amplified product to detect the target sequence.

[0074] The detection of the amplified product can be either real-time (during the amplification process) or endpoint (after the amplification process). The present invention enables the detection of the amplified product in the same container in which the amplification is carried out.

[0075] Preferably, a DNA internal control is used to monitor the amplification reaction.

[0076] Preferably, an RNA internal control is used to monitor the reverse transcription and amplification reactions.

[0077] The primers of the present invention are useful for both reverse transcription of HCirV-1 RNA and amplification of HCirV-1 DNA. The primer sequences are preferably selected for HCirV-1.

[0078] The present invention includes a set of primers, i.e., at least two primers of different orientations. Preferably, the primers are a set of one forward primer and one reverse primer. All primers referred to herein can be clearly included in this set of primers.

[0079] The "reverse primer" is an antisense primer, which can be a primer for reverse transcription, preferably does not bind to non-human circoviruses, and is specific for HCirV-1.

[0080] In one embodiment, the RNA sequence of HCirV-1 can be detected, preferably in cells, for example by ISH. In one embodiment, a cocktail of bDNA probes can be designed to target the capsid and / or Rep gene of HCirV-1, as described, for example, in the Examples. In one embodiment, the ViewRNA ISH Tissue Assay Kit 2-plex (Thermo Fisher Scientific) can be used.

[0081] Preferred DNA probes are, for example, HCirV-Pb: 5’-[FAM]GGAGATTGGAGAGAGTGGCTG[BHQ1]-3’ ([FAM]-SEQ ID NO: 15-[BHQ]) is.

[0082] Proteins and peptides The present invention encompasses proteins and peptides based on the HCirV-1 amino acid sequence. In one embodiment, the protein or peptide of the present invention is an HCirV-1 capsid or replicase protein or peptide. In one embodiment, the protein or peptide of the present invention comprises or consists of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, or 250 amino acids of SEQ ID NO: 2 (capsid protein) or SEQ ID NO: 12 (replicase protein).

[0083] In some embodiments, the protein or peptide of the present invention comprises or consists of all or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, or 250 amino acids of any of the sequences of SEQ ID NO: 2 or SEQ ID NO: 12. These peptides are referred to herein as "fragments" of the present invention.

[0084] In some embodiments, the protein or peptide of the present invention is a variant having at least 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2 or SEQ ID NO: 12.

[0085] In a preferred embodiment, the following capsid protein fragments can be used to detect the presence of the virus by serological means:

[0086] NNGTVVQKFCKTEPFTLQNQDSTNAKKWMQGSYTTTLSEWVSSPGWDFYKILKLKLTFLPFNATQRMNYWPIGNTIIDLDGPWNEPNTVQSNLLCNTATSRLWKANRKHSRYFTPRPLIHLTGNTTSSQASALLPRRTWWNSAESTVKWGGLKYSHYVEYNQPVRYWLIKSAWIKWKYLL (SEQ ID NO: 16)

[0087] NNASIVQKFCKWEPISFQNTTNQNKWYIGSYTTNLKEWVTQPNWDLYKILKMKISFLPFTPTQKMPYWPIGNTIIDLDGPWPQPYTANSNLFCNTATSRSWYGNKKHSRYFTPRAVVNLTTTATTGEGTALLPRSTWWNCNNDSIIWGGLRFAHYIENGSNMAIRYHLQKSVWIKWKYLL (SEQ ID NO: 17)

[0088] The latter sequence corresponds to a fragment of the capsid protein of human sarcovirus obtained from the second patient YN09 / J030 by the team of Li et al., Emerging Infectious Diseases, Volume 29, issued on May 5, 2023.

[0089] In some embodiments, the protein or peptide of the present invention comprises or consists of all or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, or 175 amino acids of either the sequence of SEQ ID NO: 16 or the sequence of SEQ ID NO: 17. These peptides are also referred to herein as "fragments" of the present invention.

[0090] Preferably, the protein or peptide of the present invention is labeled. In one embodiment, the protein or peptide or fragment of the present invention is labeled with a visualization molecule, such as a radioactive atom, a dye, a fluorescent molecule, a fluorophore, an enzyme, colloidal gold, magnetic particles, or latex beads.

[0091] The present invention encompasses the proteins and peptides of the present invention for use in diagnostic assays and their use in such assays.

[0092] Antibody The present invention encompasses antibodies generated against the HCirV-1 protein sequences described above. In one embodiment, the antibody binds to the protein of SEQ ID NO: 2 or SEQ ID NO: 12 or SEQ ID NO: 16 / 17. Preferably, the antibody does not bind to the capsid protein of any other circovirus such as Wolvfec circovirus (Genbank QSX73454, SEQ ID NO: 3), nor to the capsid protein of porcine circovirus 3.

[0093] The present invention encompasses the antibodies of the present invention for use in diagnostic assays and their use in such assays.

[0094] In one embodiment, the polyclonal or monoclonal antibodies of the present invention are generated in rabbits or mice.

[0095] In one embodiment, the VHH antibodies of the present invention are generated in, for example, alpacas.

[0096] The present invention encompasses polyclonal or monoclonal antibodies or fragments thereof directed against the HCirV-1 proteins or peptides or fragments described above (SEQ ID NO: 2, 12, 16, 17, or any variant or fragment thereof described above).

[0097] In one embodiment, the antibodies of the present invention can be obtained by immunizing animals with the HCirV-1 protein.

[0098] The antibodies of the present invention can function as reagents that bind to the patient's native HCirV-1 protein in immunoassays.

[0099] The antibody of the present invention can function as a positive control reagent that binds to the isolated and purified HCirV-1 protein in the immunoassay of patients.

[0100] The present invention of the present invention includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and fragments thereof (e.g., Fab, Fv, scFv) targeting the HCirV-1 protein.

[0101] For the purposes of the present invention, an expressed chimeric antibody is understood to mean an antibody that includes all or part of the heavy and / or light chains of an antibody of another animal species or another class of antibody in relation to an antibody of a specific animal species or a specific class of antibody.

[0102] In some embodiments, the purified protein is used to produce antibodies by conventional techniques. In some embodiments, recombinant or synthetic proteins or peptides are used to make antibodies by conventional techniques.

[0103] Antibodies can be synthetic, semi-synthetic, monoclonal, or polyclonal and can be produced by techniques well known in the art. Such antibodies specifically bind (not non-specifically bind) to proteins and polypeptides via the antigen-binding site of the antibody. Purified or synthetic proteins and peptides can be used as immunogens when they produce antibodies immunoreactive with them. Proteins and peptides contain antigenic determinants or epitopes that induce the formation of antibodies.

[0104] These antigenic determinants or epitopes may be either linear or structural (discontinuous). Linear epitopes are composed of a single portion of the amino acids of a polypeptide, and structural or discontinuous epitopes are composed of amino acid portions from different regions of polypeptide chains that are brought very close together by protein folding (C.A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed., 1996)). Since folded proteins have complex surfaces, the number of available epitopes is very large, but due to the three-dimensional structure and steric hindrance of the protein, the number of antibodies that actually bind to an epitope is less than the number of available epitopes (C.A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed., 1996)). Epitopes can be identified by any of the methods known in the art. Such epitopes or variants thereof can be produced using techniques well-known in the art, such as solid-phase synthesis, chemical or enzymatic cleavage of polypeptides, or recombinant DNA technology.

[0105] Antibodies are defined as specifically binding when they bind to a protein or polypeptide with a Ka of about 10 7 M -1 or greater. The affinity of a binding partner or antibody can be readily determined using conventional techniques, such as those described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).

[0106] Polyclonal antibodies can be readily generated from a variety of sources, such as horses, cows, goats, sheep, dogs, chickens, alpacas, camels, rabbits, mice, or rats, using procedures well known in the art. Generally, a purified protein or polypeptide that is appropriately conjugated is typically administered to the host animal via parenteral injection. Immunogenicity can be enhanced by the use of adjuvants, such as Freund's complete or incomplete adjuvant. After booster immunization, a small sample of serum is taken and tested for reactivity against the protein or polypeptide. Examples of various assays useful for such determinations include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, as well as procedures such as counterimmunoelectrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot assay, and sandwich assay. See U.S. Patent Nos. 4,376,110 and 4,486,530.

[0107] Monoclonal antibodies can be readily prepared using procedures well known in the art. See, for example, U.S. Patent Reissue Nos. 32,011, 4,902,614, 4,543,439, and 4,411,993; and the procedures described in Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam and Bechtol (eds.), 1980.

[0108] For example, a host animal such as a mouse can be intraperitoneally injected with an isolated and purified protein or conjugated polypeptide, such as a peptide comprising or consisting of the specific amino acids described above, at least once, preferably at intervals of about three weeks, at least twice. Subsequently, mouse sera are assayed by conventional dot blot techniques or antibody capture (ABC) to determine which animals are optimal for fusion. Approximately two to approximately three weeks later, the mice are administered an intravenous boost of the protein or polypeptide. Subsequently, according to an established protocol, the mice are sacrificed and the spleen cells are fused with a commercially available myeloma cell, such as Ag8.653 (ATCC). Briefly, the myeloma cells are washed several times in medium and fused with mouse spleen cells at a ratio of approximately three spleen cells per one myeloma cell. The fusing agent can be any suitable agent used in the art, such as polyethylene glycol (PEG). The fusions are plated onto plates containing a medium that allows for the selective growth of the fused cells. Subsequently, the fused cells can be grown for approximately eight days. The supernatant of the resulting hybridomas is recovered and added to plates first coated with goat anti-mouse Ig. After washing, a label, such as a labeled protein or polypeptide, is added to each well, followed by incubation. Subsequently, the positive wells become detectable. The positive clones can be grown in large scale culture and then the supernatant is purified by a protein A column (Pharmacia).

[0109] The monoclonal antibodies of the present invention can be produced using alternative techniques such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques that incorporate the variable regions of genes encoding specific binding antibodies. Such techniques are described in Larrick et al., Biotechnology, 7:394 (1989).

[0110] Antigen-binding fragments of such antibodies that can be produced by conventional techniques are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

[0111] The monoclonal antibodies of the present invention include chimeric antibodies, such as humanized forms of mouse monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and have the advantage of reduced immunogenicity when administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a mouse antibody (or only the antigen-binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen-binding site of a mouse monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies are described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio / Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May 1993). Procedures for generating antibodies by gene transfer can be found in GB2,272,440, U.S. Patent Nos. 5,569,825 and 5,545,806.

[0112] Antibodies produced by genetic engineering methods, such as chimeric and humanized monoclonal antibodies that contain both human and non-human parts and can be made using standard recombinant DNA techniques, can be used. Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example, International Publication No. WO87 / 02671 by Robinson et al.; European Patent Application No. 0184187 by Akira et al.; European Patent Application No. 0171496 by Taniguchi, M.; European Patent Application No. 0173494 by Morrison et al.; PCT International Publication No. WO86 / 01533 by Neuberger et al.; U.S. Patent No. 4,816,567 by Cabilly et al.; European Patent Application No. 0125023 by Cabilly et al.; Better et al., Science 240:1041 - 1043, 1988; Liu et al., PNAS 84:3439 - 3443, 1987; Liu et al., J. Immunol. 139:3521 - 3526, 1987; Sun et al., PNAS 84:214 - 218, 1987; Nishimura et al., Canc. Res. 47:999 - 1005, 1987; Wood et al., Nature 314:446 - 449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553 - 1559, 1988); Morrison, S.L., Science 229:1202 - 1207, 1985; Oi et al., BioTechniques 4:214, 1986; U.S. Patent No. 5,225,539 by Winter; Jones et al., Nature 321:552 - 525, 1986; Verhoeyan et al., Science 239:1534, 1988; and Beidler et al., J. Immunol. 141:4053 - 4060, 1988).

[0113] In the context of synthetic and semi-synthetic antibodies, such terms are intended to encompass, but are not limited to, antibody fragments, isotype-switch antibodies, humanized antibodies (e.g., mouse - human, human - mouse), hybrids, antibodies with multiple specificities, and fully synthetic antibody-like molecules.

[0114] In one embodiment, the invention encompasses single domain antibodies (sdAbs), also known as NANOBODIES. An sdAb is a fragment consisting of a single monomeric variable antibody domain that can selectively bind to a specific antigen.

[0115] In one embodiment, the sdAbs of the invention are derived from heavy chain antibodies found in camelids (VHH fragments) or cartilaginous fish (VNAR fragments), or are obtained by splitting dimeric variable domains into monomers.

[0116] Serological and virological diagnosis The HCirV-1 proteins, peptides derived from these proteins, and antibodies generated against them, as described above, can be used for the detection and diagnosis of human enterovirus infections, particularly for serological diagnosis (detection of specific antibodies) or virological diagnosis (detection of viral proteins) by immunoassay, such as immunoenzymatic methods (e.g., ELISA).

[0117] The invention encompasses a method for identifying a patient infected with enterovirus, comprising preparing a serum sample from the patient, contacting the serum with an HCirV-1 protein, and visualizing the antigen-antibody complex. Preferably, the antigen-antibody complex is visualized by EIA, ELISA, RIA, or immunofluorescence.

[0118] The invention encompasses a composition comprising an HCirV-1 protein, or the use of an HCirV-1 protein for the detection of antibodies against enterovirus in a biological sample and the diagnosis of enterovirus infections.

[0119] The antibodies and antibody fragments according to the present invention directed against the HCirV-1 protein and peptides derived therefrom are useful for the direct detection and diagnosis of circovirus infections and for the detection of HCirV-1 in biological samples. Detection of the capsid or replicase protein of HCirV-1 is carried out in biological samples taken from individuals likely to be infected, by appropriate techniques, in particular EIA, ELISA, RIA, immunofluorescence.

[0120] In one embodiment, the present invention is a method for the detection of human circovirus in a biological sample, comprising contacting a biological sample from a patient infected with circovirus with an anti-HCirV-1 antibody and visualizing the formed antigen-antibody complex. Preferably, the antigen-antibody complex is visualized by EIA, ELISA, RIA, or immunofluorescence.

[0121] In one embodiment, the HCirV-1 protein of the present invention binds to a suitable support, in particular a microplate or beads.

[0122] In one embodiment, the method of the present invention comprises contacting a biological sample from a subject, preferably a human, infected with circovirus with an HCirV-1 protein bound to a suitable support, in particular a microplate or beads, to effect binding, washing the support to remove unbound antibody, adding a detection reagent that binds to the immunoglobulin bound to the HCirV-1 protein, and detecting the formed HCirV-1 protein-antibody complex.

[0123] In one embodiment, the method of the present invention for the detection of antibodies against circovirus in a biological sample comprises providing an HCirV-1 protein, providing a biological sample from a patient infected with circovirus, contacting the HCirV-1 protein with the biological sample, and visualizing the formed antigen-antibody complex. Preferably, the method comprises ELISA.

[0124] Preferably, the protein-antibody complex is detected using an antibody or antibody fragment that binds to human immunoglobulins.

[0125] Preferably, the detection reagent comprises a label selected from chemiluminescent labels, enzymatic labels, fluorescent labels, and radioactive (e.g., iodine) labels. Most preferably, the detection reagent is a labeled antibody or antibody fragment that binds to human immunoglobulins.

[0126] Preferred labels include fluorescent labels such as FITC, chromophore labels, affinity ligand labels, enzymatic labels such as alkaline phosphatase, horseradish peroxidase, luciferase, or galactosidase, enzyme cofactor labels, hapten conjugate labels such as digoxigenin or dinitrophenyl, Raman signal generating labels, magnetic labels, spin labels, epitope labels such as FLAG or HA epitopes, luminescent labels, heavy atom labels, nanoparticle labels, electrochemical labels, light scattering labels, spherical shell labels, semiconductor nanocrystal labels, where the label can enable visualization with or without using a secondary detection molecule.

[0127] Preferred labels include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, or acetylcholinesterase; elements of binding pairs capable of forming a complex such as streptavidin / biotin, avidin / biotin, or antigen / antibody complexes including for example rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, eosin, green fluorescent protein, erythrosin, coumarin, methylcoumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes including for example europium and terbium, members of the cyanine dye family such as Cy3 and Cy5, molecular beacons and their fluorescent derivatives, and others known in the art; luminescent substances such as luminol; light scattering or plasmon resonance materials such as gold or silver particles or quantum dots; or 14 C, 123 I, 124 I, 125 I, 32 P, 33 P, 35 S, or 3 radioactive substances containing C, I, I, I, P, P, S, or H.

[0128] In one embodiment, the antibody or antibody fragment of the invention that binds to human immunoglobulins specifically binds to IgG, IgA, and IgM. In one embodiment, the antibody or antibody fragment of the invention that binds to human immunoglobulins specifically binds to IgG, IgA, or IgM.

[0129] The term "antibody" is intended to include polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab’)2 and Fab fragments, single-chain variable fragments (scFv), single-domain antibody fragments (VHH or nanobodies), diabody fragments, and any binding partners produced recombinantly and synthetically.

[0130] In a preferred embodiment, the antibody of the present invention is a VHH, preferably alpaca serum.

[0131] In one embodiment, the method of the present invention includes comparing the results obtained using patient serum with positive and negative controls.

[0132] As positive controls, - sera from animals (e.g., rabbits, alpacas, etc.) immunized with the HCirV-1 protein described above, - the HCirV-1 protein described above, preferably including SEQ ID NO: 2 can be mentioned.

[0133] The method of the present invention can include the use of the HCirV-1 protein for detecting a novel circovirus that does not cross-react with circoviruses of other species.

[0134] In one embodiment, the method of the present invention includes an immunocapture method.

[0135] In one embodiment, the method of the present invention includes binding a first monoclonal or polyclonal antibody or fragment thereof (capture antibody) targeting the HCirV-1 protein, incubating the antibody with a biological sample containing the HCirV-1 protein, and detecting the formed antigen-antibody complex, preferably using a monoclonal antibody (visualization antibody).

[0136] In one embodiment, in the method of the present invention, the biological sample used is mixed with a visualization monoclonal antibody before being contacted with the capture antibody.

[0137] In the immunocapture assay according to the present invention, it is possible to use a monoclonal antibody conjugated with a visualization molecule or particle to visualize the HCirV-1 protein.

[0138] The visualization molecule can be a radioactive atom, a dye, a fluorescent molecule, a fluorophore, an enzyme, and the visualization particle can be, for example, colloidal gold, magnetic particles, or latex beads.

[0139] The subject of the present invention is also an immune complex formed from the polyclonal or monoclonal antibody or antibody fragment defined above, and the HCirV-1 protein.

[0140] Antigen and antibody detection kit The present invention encompasses a circovirus detection kit characterized by containing the HCirV-1 protein described above and / or an antibody produced against them.

[0141] In one embodiment, the HCirV-1 protein of the present invention contains the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 12 or SEQ ID NO: 16 / 17 described above, or a fragment or variant thereof.

[0142] In one embodiment, the present invention includes a kit for the detection of circovirus infection, which kit contains the HCirV-1 protein and reagents described above for the detection of antigen-antibody complexes.

[0143] Preferably, the kit of the present invention contains the serum of an animal immunized with the HCirV-1 protein.

[0144] Most preferably, the serum of the present invention is rabbit or alpaca serum derived from an animal immunized with the HCirV-1 protein.

[0145] In one embodiment, the kit of the present invention comprises an HCirV-1 protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 12 or SEQ ID NO: 16 or SEQ ID NO: 17 as described above, or a fragment or variant thereof.

[0146] In one embodiment, the kit of the present invention comprises both an HCirV-1 protein and an HCirV-1 immune serum.

[0147] In one embodiment, the kit of the present invention is a simple / rapid test designed for use when preliminary screening test results are required. The test can be a test based on agglutination, immunodot, immunochromatography and / or immunofiltration techniques. Preferably, the test is performed in a short time and easily, preferably in about 10 minutes to about 2 hours, and requires little or no additional equipment.

[0148] Preferably, the kit of the present invention can be stored at room temperature for a long period of time.

[0149] Immunogenic composition The present invention also relates to an immunogenic composition or vaccine composition comprising a peptide or protein according to the present invention, such as SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16 or SEQ ID NO: 17, or any variant / fragment thereof.

[0150] In certain embodiments, the immunogenic composition of the present invention further comprises an adjuvant and / or a pharmaceutically acceptable vehicle.

[0151] As defined herein, a pharmaceutically acceptable vehicle includes any substance that enables the formulation of the polyepitopes, polynucleotides, and vectors according to the present invention in a composition. The vehicle is any substance or combination of substances that is physiologically acceptable, i.e., suitable for use in a composition that contacts a host, especially a human, and is thus non-toxic. Examples of such vehicles are phosphate-buffered saline aqueous solutions, distilled water, emulsions such as oil / water emulsions, various types of wetting agents, and sterile solutions.

[0152] As defined herein, an adjuvant can include, for example, liposomes, an oil phase commonly used in the form of an emulsion with an aqueous phase, such as Freund's type adjuvants, or can include water-insoluble inorganic salts such as aluminum hydroxide, zinc sulfate, iron hydroxide colloid, calcium phosphate, or calcium chloride.

[0153] In another specific embodiment, the immunogenic composition of the present invention is formulated for administration by a parenteral route, such as subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.), intraperitoneal (i.p.), or intravenous (i.v.) injection.

[0154] In another specific embodiment, the immunogenic composition of the present invention is administered in one or more doses, especially in a prime-boost dosing schedule.

[0155] The amount (dose) administered depends on the subject being treated, e.g., the condition of the patient, the state of the individual's immune system, the route of administration, and the size of the host. Suitable doses range from 10 3 TCID50 to 10 7 TCID50, or in the range of 100 micrograms of plasmid DNA, and can be varied by those skilled in the art depending on the circumstances.

[0156] In a preferred embodiment, the immunogenic composition or vaccine composition of the present invention is for use in the prevention of sarcovirus infections and related diseases, particularly hepatitis, in human subjects.

Examples

[0157] We report the case of a 61-year-old female who had received a solid organ transplant and presented with hepatolytic hepatitis of unknown etiology. The patient had received a heart-lung transplant 17 years ago due to Eisenmenger syndrome associated with ventricular septal defect. The maintenance immunosuppressive regimen included tacrolimus, mycophenolate mofetil, and prednisone. She had received pulse steroids 16 months before the onset of hepatitis due to acute transplant lung dysfunction. The patient had a history of surgically treated localized left upper lobe lung adenocarcinoma 2.5 years before presentation. She also had a history of chronic kidney disease (23 mL / min by Cockcroft-Gault) due to calcineurin inhibitor, bronchopulmonary infections due to Aspergillus and Scedosporium species, parvovirus B19 infection, and COVID-19 infection.

[0158] In November 2021, the patient was admitted for CMV colitis with resistant virus, requiring foscarvir and subsequent maribavir treatment, followed by long-term maintenance therapy with letermovir. At this time, cytolysis without symptoms or liver abnormalities was detected by ultrasound (Figure 4).

[0159] Four months after this hospitalization, her hepatitis worsened, with serum transaminase levels rising up to 40 times the upper limit of normal (Figure 4), leading to readmission. This patient was asymptomatic except for recent weight loss and had normal prothrombin time levels, so no biological severity was recognized. Usual infectious diseases including hepatitis A, B, C, E, HIV, CMV, HSV, VZV, human herpesvirus 6, adenovirus, enterovirus (in blood and stool), parvovirus B19, toxoplasmosis, syphilis, and leptospirosis were excluded. Only EBV PCR was positive (104.4 genome copies / mL blood). There was no evidence regarding the temporal relationship between the changes in liver enzymes and the various medications of this patient. She reported no use of paracetamol, alcohol, illegal drugs, or phytotherapy. Echocardiogram showed no heart failure. No cardiac arrhythmia, vascular or biliary abnormalities were present on liver ultrasound and Doppler examination, CT scan, and magnetic resonance cholangiopancreatography (MRCP). Tests for autoimmune abnormalities were negative. Finally, aceruloplasminemia was normal. Then, a liver biopsy was performed. Pathological examination showed slightly inflammatory lobular hepatitis without epithelioid granulomas, marked portal inflammation and fibrosis, or lymphoproliferation. Ziehl, EBV, CMV, and adenovirus staining were negative. Microbiological examination of the biopsy specimen (including mycobacterium culture and PCR, broad-range 16S ribosomal RNA gene PCR) was negative. HSV-1, -2, CMV, adenovirus, and enterovirus PCR were negative, and EBV PCR was positive (135 gc / g DNA, cycle threshold = 35), but acute hepatitis was not considered the cause. Overall, the lymphocytic pattern of inflammation strongly suggested viral hepatitis, in contrast to the absence of eosinophil infiltration, vascular or biliary injury, and autoimmune liver disease (without plasma cells). The degree of both centrilobular necrosis and reabsorption by macrophages suggested recent significant hepatocyte lysis. After these negative results for first-choice tests, metagenomic next-generation sequencing (mNGS) was performed on the liver biopsy, which showed a novel circovirus (HCirV-1).Further exploration and quantification of HCirV-1 by qPCR were performed on liver tissue, stool, bronchoalveolar lavage fluid, urine, and saliva (Table 1). Examination and testing of the patient's past blood samples were also conducted (Table 2).

[0160]

Table 1

[0161]

Table 2

[0162] As of June 2022, the patient's clinical condition is currently stable. The inventors observe an improvement in cell lysis. In the context of this recent infectious disease, immunosuppressive treatment has been tapered down to just minimal mycophenolate mofetil (Figure 4). The inventors attempted to identify potential sources of HCirV-1 infection. The patient reported contact with two cats, both of which had contact with birds and rodents. Two months after diagnosis, HCirV-1 PCR of her cat's feces was negative. The patient reported no foreign travel. She reported a blood transfusion performed 17 months prior to the initial HCirV-1 detection (Figure 4).

[0163] The inventors searched for viral sequences in the liver of a heart-lung transplant recipient presenting with severe liver cell lysis and identified a new circovirus.

[0164] Damaged liver tissue was obtained from a biopsy and subjected to ultra-deep sequencing (high-throughput sequencing, or NGS).

[0165] Nucleic acids from the liver biopsy of this patient were extracted using the geneLEAD VIII device (Diagenode), and a concentration of 15 ng / L was obtained. The RNA Integrity Number (RIN) after extraction was 2.9. For metagenomic next-generation sequencing library preparation, two protocols were followed: (i) A cDNA library was constructed using the SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian (Takara Bio Inc., Kit v.3). The procedure included random reverse transcription of total RNA into first-strand cDNA, depletion of human ribosomal cDNA, and final PCR amplification; (ii) The SuperScript IV First-Strand Synthesis System kit (Invitrogen) was used with random primers according to the manufacturer's instructions, followed by MALBAC amplification (Yikon Genomics) to construct a cDNA library, which was used as input for Illumina DNAPrep (Illumina, formerly Nextera DNA Flex) to generate the final NGS library.

[0166] The RT / MALBAC / DNA Prep library was sequenced at 1×151 bp on a NextSeq500 device (Illumina) using a High Output flow cell, generating approximately 80 million raw reads. Most of the raw reads had a quality score (Q-score) of over 20 (Figure 2).

[0167] Raw reads from enrichment-based sequencing were processed using an in-house bioinformatics pipeline (Microseek; Bigot et al., DOI: 10.3390 / v14091990) that included quality checking and trimming, read normalization, de novo assembly, and ORF prediction for contigs and singletons, followed by three levels of taxonomic assignment. The resulting viral hits with their associated taxonomic levels were shown as Krona (Figure 3).

[0168] The longest capsid contig available (k141_422_flag=1_multi=91.0000_len=931_742_41__46_234) has 39% identity at the amino acid level to the most closely related virus identified, the Wolvfec circovirus (Genbank QSX73454). The sequences identified as Circoviridae were then mapped to appropriate reference sequences using GENEIOUS PRIME 2022.1.1 (Biomatters Ltd) to describe the complete genome (Figure 3). The complete sequence of the capsid was reconstructed from the longest capsid contig used as the species sequence using an iterative mapping approach. The nucleotide and amino acid sequences of the reconstructed capsid are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0169] A novel circovirus species was identified and named Human circovirus 1 (HCirV-1) within the genus Circovirus of the family Circoviridae. Pathogenic circoviruses have not been previously known in humans.

[0170] A phylogenetic tree was constructed based on the sequences and is shown in Figure 1. Phylogenetic analysis of the capsid gene (blast.ncbi.nlm.nih.gov / ) indicates that this virus corresponds to a new clade within the members of the genus Circovirus.

[0171] The capsid of the novel human circovirus shows only 39% identity at the amino acid level to the most closely related circovirus, the Wolvfec circovirus, i.e., Genbank QSX73454 shown below:

[0172] MRRYRRLRRR RRRRPRRPRY RRRIRRTWIR RPTAGTYYTK KYSTMNIINL GKRESTKYWKVGHYTTSLKE WGSKWIWDYY KILKMKITFY PQESNCFQHS LLWGHTVIDY DGSWSTEGWLQDDPYANSST GRVWMSNKKH SRYFTPKPFL VQTSSSYTGQ SLFYFTRNTP WLNCYDLDTKWGALLFSAYA PADDTTPLYV QKSVWIRFKT VL(SEQ ID NO: 3)

[0173] In contrast, in animals, there are a number of known circoviruses, including those suspected of being liver-directed viruses (Equine sarcoidosis virus strain 1). (Beak and feather disease virus (BFDV), Porcine circovirus 1, 2, and 3 (PCV1-3)). Hui et al., 2021 (https: / / doi.org / 10.3390 / v13050944). The nucleotide and amino acid sequences of the capsid protein of HCirV-1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0174] The number of viral sequences in liver biopsies was very high (more than 1000 contigs and singletons), and no other viral sequences or bacterial sequences were detected by NGS, supporting the role of HCirV-1 in this disease.

[0175] Explanation of the control The inventors retrospectively performed control HCirV-1 qPCR on stool and blood samples from immunocompromised or immunocompetent patients with or without hepatitis (Table 3). The inventors also examined the results of mNGS, which has been regularly performed on liver biopsies for pathogen diagnosis since 2019 in their laboratory (Table 4).

[0176]

Table 3

[0177]

Table 4

[0178] Nucleic acid extraction Nucleic acids from the liver biopsy of this patient were extracted using a geneLEAD VIII device (Diagenode). Nucleic acids from 200 μL of plasma, serum, whole blood, bronchoalveolar lavage fluid (BAL), and feces were extracted using an Emag device (Biomerieux).

[0179] mNGS cDNA was generated from the liver biopsy of this patient using the SuperScript IV First-Strand Synthesis System kit (Invitrogen). MALBAC amplification (Yikon Genomics) was used as input for Illumina DNAPrep (Illumina, formerly Nextera DNA Flex) as previously described [Regnault B, Bigot T, Ma L, Perot P, Temmam S, Eloit M. Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results. Viruses 2021;13:253]. Libraries were sequenced at 1×151 bp on a NextSeq500 device (Illumina), generating approximately 80 million raw reads. The raw reads were processed using an in-house bioinformatics pipeline using the RVDB-prot reference viral database. Bigot T, Temmam S, Perot P, Eloit M. RVDB-prot, a reference viral protein database and its HMM profiles. F1000Res 2019;8:530.

[0180] qPCR HCirV-1 The following PCR primers were designed for the capsid gene of HCirV-1: HCirV1-Fw1: 5-ACCTGGATGGACCCTGGAAT-3 (SEQ ID NO: 4); HCirV1-Rv1: 5-AGAGTTCCACCAGGTTCTGC-3 (SEQ ID NO: 5) (194 bp). Quantitative PCR was performed in the SYBR Green format with 45 cycles of amplification and an annealing temperature of 58 °C. The positive amplicons after 45 cycles were purified on a gel, confirmed by Sanger sequencing, and used for serial dilution to create a standard curve for calculating the virus amount.

[0181] Acquisition of the complete genome The complete genome of HCirV-1 was obtained by sequencing the plasma of this patient on January 12, 2022 (containing 1010 virus copies / mL) after pre-amplification using phi29 polymerase (WTA, Qiagen). A final assembly was performed by applying an iterative mapping method using Geneious Prime 2022.1.1 (Biomatters Ltd). The complete genome sequence was deposited in GenBank under the accession number ON677309.

[0182] Viral sequences were analyzed from liver biopsy specimens using the identification of novel circoviruses in liver biopsy NGS. A total of 1011 sequences of the family Circoviridae corresponding to the capsid and Rep genes were identified. From plasma samples collected at the peak of viremia, the inventors derived the consensus complete genome sequence of the virus (2021 pb), named this HCirV-1 (Genbank ON677309), and confirmed that the sequence was identical to the partial sequence of the capsid gene found in the liver. The inventors performed phylogenetic analysis on two viral proteins, namely the capsid and Rep of HCirV-1, and representative animal circoviruses. The two analysis results were consistent, indicating that HCirV-1 defines a new clade at a position close to the base compared to the most closely related virus species (Wolvfec circovirus and porcine circovirus 3).

[0183] Systematics Phylogenetic tree reconstruction was performed for the capsid protein sequence, replicase (Rep) protein sequence, and complete nucleotide sequence of HCirV-1. The complete ORFs of the capsid and replicase genes were aligned with other representative sequences of circoviruses using the MAFFT (Multiple Alignment using Fast Fourier Transform) alignment tool with the LINS-i parameter. Phylogenetic tree reconstruction by the maximum likelihood method was performed using PhyML implemented via the NGPhylogeny portal. Lemoine F, Correia D, Lefort V et al., NGPhylogeny.fr: new generation phylogenetic services for non-specialists. Nucleic Acids Research 2019;47:W260~W265. The support rate of the nodes was evaluated using "Approximate Bayes branch support".

[0184] TTV and EBV qPCR The measurement of TTV DNA amount was performed as previously described using the TTV R-GENE (registered trademark) kit (Biomerieux, Marcy-l’Etoile, France). Kulifaj D, Durgueil-Lariviere B, Meynier F et al., Development of a standardized real time PCR for Torque teno viruses (TTV) viral load detection and quantification: A new tool for immune monitoring. J Clin Virol 2018;105:118~127. The measurement of EBV DNA amount was performed using the EBV R-GENE (registered trademark) kit (Biomerieux, Marcy l’Etoile, France). Real-time PCR amplification was performed on the AB7500 platform (Applied Biosystems, Waltham, MA, United States).

[0185] The inventors designed and performed quantitative PCR (qPCR) to evaluate changes in the amount of virus in plasma and other biological samples collected from this case since 2017. The inventors also evaluated the amount of torque teno virus (TTV), a commensal virus whose viremia level correlates well with the immune status of transplant recipients. Jaksch P, Goerzer I, Puchhammer-Stoeckl E, Bond G. Integrated Immunologic Monitoring in Solid Organ Transplantation: the Road Towards Torque Teno Virus-guided Immunosuppression. Transplantation 2022. The amount of TTV DNA fluctuated from 0 to 3.99 log cp / ml from 2017 to the present. This indicates a low level of immunosuppression [Redondo N, Navarro D, Aguado JM, Fernandez-Ruiz M. Viruses, friends, and foes: The case of Torque Teno Virus and the net state of immunosuppression. Transpl Infect Dis 2022;24:e13778], and the peak of TTV viremia was not associated with the peak in cytolysis. In contrast, the first presence of HCirV-1 in the blood may have been in September 2020 (103.3 gc / mL), which was 2 months after the steroid pulse, but the sample immediately preceding this (July 2020) was negative. Nevertheless, it is noteworthy that this low level of HCirV-1 in September 2020 was suspected because the tube was opened simultaneously with other tubes that carried up to 109.1 gc / mL, and cross-contamination may have occurred. The next available sample was collected in September 2021 and was strongly positive (107.5 gc / mL). Thereafter, the amount fluctuated between 108.3 and 1010.2 gc / mL. The amount of HCirV-1 was high only during periods of significant cytolysis.

[0186] Evaluation of the presence of HCirV-1 in controls In controls tested using the same specific qPCR primers to assess the presence of HCirV-1, no positive samples were detected. A total of 36 blood samples and 20 stool samples from 56 patients were tested (Table 3). Of these patients, 19 (34%), 17 (30%), 13 (23%), 5 (9%), and 2 (4%) were patients with congenital immunodeficiency, immunocompetent patients, solid organ transplant recipients, hematopoietic stem cell transplant recipients or patients receiving immunosuppressive treatment, and HIV-positive patients, respectively. Of the 16 patients with hepatocyte lysis (Table 3), 8 had an unknown etiology. Control biopsies that have been regularly screened by mNGS since 2019 showed no HCirV-1 sequences in 57 patients with hepatitis and suspected sources of infection, and 25 (44%), 20 (35%), 5 (9%), 5 (9%), and 2 (4%) were patients with congenital immunodeficiency, hematopoietic stem cell transplant recipients or patients receiving immunosuppressive treatment, immunocompetent patients, solid organ transplant recipients, and HIV-positive patients, respectively (Table 4). The inventors here show the temporal association between a large amount of novel human virus (HCirV-1) in the liver and blood and hepatitis in a 61-year-old female with a heart-lung transplant. This patient received a standard immunosuppressive regimen, and TTV viremia did not support a high level of immunosuppression. These results should be evaluated in the context of Hill's criteria for causation. Hill AB. The Environment and Disease: Association or Causation? Proc R Soc Med 1965;58:295~300. Specificity of the association was demonstrated by the absence of any other virus detected by PCR, except EBV, using comprehensive mNGS in liver biopsies. Considering the low EBV concentration in liver biopsies, simultaneous higher replication in blood (104.4 genome copies / mL), and negative results of EBV staining on liver biopsies, EBV was not considered to be the cause of such cytolysis.HCirV-1 was not demonstrated by qPCR in 40 controls without hepatitis, nor in 16 controls with hepatitis of known (50%) or unknown (50%) etiology (Table 3). Additionally, HCirV-1 was not detected in 57 additional cases of hepatitis of unknown etiology at the time of prescription that were investigated by mNGS (unpublished results, Table 4). In summary, the inventors did not detect the HCirV-1 genome in 113 controls.

[0187] Circoviruses are DNA viruses. Importantly, HCirV-1 transcripts were detected by specific ISH in the nuclei of 2% of liver cells. Thus, the ISH results were not inconsistent with viral expression and perhaps viral replication. Considering the lytic cycle of circoviruses, this result strongly supports a role of HCirV-1 as a cause of hepatitis by targeting and lysing hepatocytes, as well as a causative role as a trigger that can induce an immunocytotoxic response.

[0188] Animal circoviruses such as porcine circovirus (PCV) 1-4 can cause reproductive disorders, dermatitis, nephropathy, and respiratory diseases that were not observed here [Sirisereewan C, Thanawongnuwech R, Kedkovid R. Current Understanding of the Pathogenesis of Porcine Circovirus 3. Pathogens 2022;11:64; Wang D, Mai J, Yang Y, Xiao C-T, Wang N. Current knowledge on epidemiology and evolution of novel porcine circovirus 4. Veterinary Research 2022;53:38]. The association between circovirus and hepatitis has been demonstrated in horses [Hui A, Altan E, Slovis N, Fletcher C, Deng X, Delwart E. Circovirus in Blood of a Febrile Horse with Hepatitis. Viruses 2021;13:944]. After inoculation of PCV3 into piglets, viremia gradually increased up to a maximum of 108.9 copies / mL, suggesting the findings of the present inventors, but overall reached a peak approximately 21 days post-infection (dpi), contrary to the later peak identified by the present inventors.Pathological lesions and PCV3 antigen were detected in the liver (including the presence of white-gray nodules and necrosis, and PCV-3 antigen was detected in the hepatic lobular stroma, hepatic sinus antral wall, and the cytoplasm of the sinusoids (Fujiwara K, Kojima H, Yasui S et al., Hepatitis A viral load in relation to severity of the infection. Journal of Medical Virology 2011;83:201~207)) and other organs (lung, heart, kidney, lymph node, spleen, and small intestine) [Jiang H, Wang D, Wang J et al., Induction of Porcine Dermatitis and Nephropathy Syndrome in Piglets by Infection with Porcine Circovirus Type 3. Journal of Virology 2019;93:e02045]. Interestingly, PCV3 viremia could still be observed at 140 dpi [Opriessnig T, Prickett JR, Madson DM et al., Porcine circovirus type 2 (PCV2)-infection and reinoculation with homologous or heterologous strains: virological, serological, pathological and clinical effects in growing pigs. Vet Res 2010;41:31], which indicates the ability to sustain a long-term infection of circovirus as described for this patient. Regarding validity, there was no temporal association for either drug etiology or pathological evidence. General hepatitis virus infections were excluded. EBV was positive, but the EBV viral load was stable at approximately 104 gc / ml (Figure 4), and EBER staining was negative, so the role of EBV as a pathogen was excluded.The first detection of HCirV-1 was before liver pathology, and more precisely, the first and second viral load peaks were each before the first and second cytolysis peaks, and the third viral load peak was simultaneous with the cytolysis peak (Figure 4). High viral load of HCirV-1 (1010 gc / ml) also supports the role of HCirV-1 as a cause of hepatitis. Indeed, a strong relationship between viral load and disease severity has been described for HBV [Yu SJ, Kim YJ. Hepatitis B viral load affects prognosis of hepatocellular carcinoma. World J Gastroenterol 2014;20:12039~12044], HAV [Fujiwara K, Kojima H, Yasui S et al., Hepatitis A viral load in relation to severity of the infection. Journal of Medical Virology 2011;83:201~207], or adenovirus in hematopoietic stem cell transplant recipients [Lion T, Baumgartinger R, Watzinger F et al., Molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease. Blood 2003;102:1114~1120]. Thus, viral load affects organ pathology [Funk GA, Gosert R, Hirsch HH. Viral dynamics in transplant patients:implications for disease. The Lancet Infectious Diseases 2007;7:460~472]. At present, the inventors have not identified the source of infection. Specifically, it is unclear whether it is due to spillover of an animal virus or whether this is the first identified case of a human virus that has become apparent.HCirV-1 has never been identified as part of the human virome before. The inventors predict that it may be of animal origin and possibly foodborne, similar to hepatitis E virus. Phylogenetic analysis positioned this virus far from the closest known viruses, which themselves infect wild exotic animals that could not be the cause in this patient. Development of specific antibody tests will help elucidate the virus's ecology and better understand the infection mechanism.

[0189] As shown in Table 2, HCirV-1 is excreted in saliva, urine, and feces and may thus potentially infect contacts, although this has not yet been investigated.

[0190] in situ hybridization Using the ViewRNA ISH Tissue Assay Kit 2-plex (Thermo Fisher Scientific), the RNA sequence of HCirV-1 as a feature of virus replication was detected. Cocktails of 12 and 20 custom-designed branched DNA (bDNA) probes were designed to target the capsid and Rep genes of HCirV-1, respectively, which were indicated by a red signal (as "probe type 1" of the kit). A mix of control probes targeting human GAPDH, ACTB, and PPI transcripts was indicated by a blue signal (as "probe type 6" of the kit). To expose the RNA target, tissue sections (after deparaffinization using xylene) were pretreated by 20 min of heat pretreatment and 15 min of protease digestion. Harris hematoxylin was used for counterstaining. Negative liver biopsy controls used for ISH included a negative control without a probe from a patient infected with HCiV-1 recommended by the manufacturer and negative controls from different patients with negative test results for HCirV-1 using metagenomic next-generation sequencing (mNGS) (the entire procedure assay was performed on this slide).

[0191] In situ hybridization (ISH) showed 2% infected (red signal) hepatocytes in the liver biopsy (Figure 7). The red labeling was strong in the nucleus and weak in the cytoplasm. Some hepatocytes had insufficient red labeling in the nucleus, and the red labeling was negative in the cytoplasm. Endothelial cells and lymphocytes were not infected. No red signal was recorded in the negative control. Detection of HCirV-1 expression in liver cells by ISH, exclusion of other hepatitis etiologies, and the temporal association of severe cytolysis with a very high amount of HCirV-1 in the liver and blood strongly suggest a causal relationship. Furthermore, the inventors' experience using mNGS in immunocompromised patients (including transplant recipients) and specific qPCR performed on a representative cohort of these patients demonstrate that HCirV-1, unlike TTV, is not part of the commensal virome.

Claims

1. Recombinant nucleic acid comprising a human circovirus 1 (HCirV-1) nucleotide sequence, wherein the sequence is preferably sequence number 10 or a variant thereof.

2. The recombinant nucleic acid according to claim 1, wherein the nucleotide sequence is an HCirV-1 capsid or replicase sequence.

3. The recombinant nucleic acid according to claim 1, wherein the nucleotide sequence comprises the capsid nucleotide sequence of SEQ ID NO: 1 or the replicase nucleotide sequence of SEQ ID NO: 11, or a variant thereof or a fragment of at least 10 consecutive nucleotides thereof.

4. The recombinant nucleic acid according to claim 1, wherein the nucleotide sequence encodes a protein having at least 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO:

17.

5. In vitro use of isolated HCirV-1 nucleic acid as a diagnostic reagent, wherein the nucleic acid preferably comprises the nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 1, or SEQ ID NO: 11, or a fragment of at least 10 consecutive nucleotides thereof.

6. A primer or probe comprising at least 10 consecutive nucleotides from the sequence of SEQ ID NO: 1, or from either SEQ ID NO: 10 or SEQ ID NO:

11.

7. The primer or probe according to claim 6, having a sequence selected from sequence numbers 4 to 9 or sequence numbers 13 to 15, and preferably labeled with a fluorescent label, radioactive label, or enzyme label.

8. A kit comprising the primer or probe described in claim 6 and an amplification and / or hybridization reagent.

9. In vitro use of the recombinant nucleic acid described in claim 1, the primer or probe described in claim 6, or the kit described in claim 8 in a diagnostic assay.

10. An in vitro method for detecting the HCirV-1 virus, wherein the method comprises contacting a primer or probe according to claim 6 with a biological sample, and detecting the presence or absence of HCirV-1 nucleic acid in the sample.

11. In vitro use of an isolated HCirV-1 protein or peptide as a diagnostic reagent, wherein the protein or peptide preferably has SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 17, or a fragment or variant thereof.

12. A pharmaceutical composition comprising an isolated HCirV-1 protein or peptide for use as a vaccine in the prevention of circovirus infections and related diseases, particularly hepatitis, in human subjects, wherein the protein or peptide preferably has SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 17, or a fragment or variant thereof.

13. An isolated protein or peptide containing at least 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acids of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 17, or a variant thereof.

14. In vitro use of isolated proteins or peptides containing at least 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acids of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 17, or their variants, as diagnostic agents.

15. A pharmaceutical composition comprising an isolated protein or peptide for use as a vaccine in the prevention of circovirus infections and related diseases, particularly hepatitis, in human subjects, wherein the protein or peptide comprises at least 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acids of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, or variants thereof.

16. An isolated antibody that specifically binds to the HCirV-1 protein or peptide, wherein the protein or peptide preferably has SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 17, or a fragment or variant thereof.

17. The isolated antibody according to claim 16, wherein the protein has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 17, or a fragment or variant thereof.

18. In vitro use of HCirV-1 virus protein as an antigen in a diagnostic assay, wherein the protein preferably has SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 17, or a fragment or variant thereof.

19. A pharmaceutical composition comprising the HCirV-1 virus protein for use as an antigen in a vaccine for the prevention of circovirus infections and related diseases, particularly hepatitis, in human subjects, wherein the protein preferably has SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 17, or a fragment or variant thereof.

20. An in vitro method for detecting HCirV-1, comprising contacting a biological sample with the protein or peptide described in claim 13 or the isolated antibody described in claim 16, and detecting the presence or absence of the HCirV-1 protein or antibody in the sample.

21. An immunogenic composition or vaccine composition comprising the protein or peptide described in claim 13.

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