Method for predicting COVID-19 infectiousness

Abstract

A method for predicting whether a subject infected with SARS-CoV-2 is likely to be infectious through airborne transmission or not is provided. The method includes determining expression of the TBLIXRI gene and optionally also the MIR4323 gene, the PPP3CB gene and the TRPM2 gene in a biological sample from a subject, typically from a subject who has tested positive for COVID-19. The method can include determining the expression levels of combinations of two or three of these genes, for example TBLIXRI and PPP3CB; TBLIXRI and MIR4323; TBLIXRI and TRPM2; TBLIXRI, PPP3CB and TRPM2; TBLIXRI, MIR4323 and PPP3CB; or TBLIXRI, MIR4323 and TRPM2. The level(s) of expression of the gene(s), through the quantification of mRNA level(s), can be compared to reference levels (non-infectious control subjects) to determine whether the subject is likely to be infectious. The biological sample is typically a blood sample. Also claimed are primer pairs for amplifying the genes.

Description

FIELD OF THE INVENTION Biomarkers for predicting the infectiousness of COVID-19 in a subject are described herein. BACKGROUND TO THE INVENTION Coronavirus disease 2019 (COVID-19) is a contagious disease caused by the virus SARS-CoV-2. COVID-19 has resulted in over 7 million recorded deaths over the last four years. In addition to the impact of these deaths on families and communities, the COVID-19 pandemic has led to severe economic hardship for many individuals, companies and even countries. Although the disease severity and mortality of COVID-19 have decreased it is still a substantial cause of mortality and severe illness in the elderly and other vulnerable groups, and in countries mortality is still significant. SARS-CoV-2 transmits when infectious particles are breathed in or come into contact with the eyes, nose, or mouth. Apart from vaccination, the best way to reduce transmission of the virus is for symptomatic individuals to self-isolate, and currently self-isolation periods of 5 to 7 days are generally advised. However, people remain contagious for up to 20 days and can spread the virus even if they do not develop symptoms, and at least a third of people who are infected do not develop noticeable symptoms. Also, many people who experience COVID-19 symptoms might not actually have COVID-19. In addition, although COVID-19 tests have been developed to detect the virus's nucleic acid in samples (including real-time reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification, and reverse transcription loop-mediated isothermal amplification (RT-LAMP) from a nasopharyngeal swab), the detection of the virus is not an indicator itself that the patient is infectious (as such tests may be positive for several months). Thus, many infectious people do not self-isolate or do not self-isolate fora long enough period, with the result that they are likely to transmit the virus to others. Conversely, many people who self-isolate may not actually be infectious and this unnecessary self-isolation may have negative economic and social consequences (such as unnecessary time off work, missing school or academic lessons, lack of care from caregivers who are self-isolating, etc.). Furthermore, if there are elderly or vulnerable persons in a household that may have a higher risk of mortality from COVID-19, or in a facility (e.g. nursing home or hospital), it would be valuable to identify infectious persons with COVID-19 (even if that specific person is not at a higher risk of mortality or morbidity). It would therefore be useful to have a test, in particular a same-day test, which can inform people of whether they are infectious or not. SUMMARY OF THE INVENTION According to a first embodiment of the invention, there is provided a method of predicting COVID-19 infectiousness, the method comprising: determining expression of the TBL1XR1 gene in a biological sample from a subject having or possibly having COVID-19; optionally also detecting expression of at least one of the PPP3CB, TRPM2 and MIR4323 genes; wherein the level of expression of TBL1XR1 or at least one of the other genes indicates the likely infectiousness of the subject. The expression of TBL1XR1 and PPP3CB may be determined. The expression of TBL1XR1 and MIR4323 may be determined. The expression of TBL1XR1 and TRPM2 may be determined. The expression of TBL1XR1, PPP3CB and TRPM2 may be determined. The expression of TBL1XR1, PPP3CB and MIR4323 may be determined. The expression of TBL1XR1, MIR4323 and TRPM2 may be determined. The sample may be a blood sample. The sample may be from a subject who has tested positive for COVID-19. The expression of the gene(s) may be determined by determining the mRNA level of the gene(s) in the sample. The expression of the gene(s) may be determined by performing PCR. The gene expression in the sample may be compared with a reference level of the same gene(s) in order to determine whether the subject is infectious. According to a second embodiment of the invention, there is provided a primer pair for use in amplifying the TBL1XR1 gene, the primer pair comprising: Forward primer: TCATCCTAAGTGCTGGAGTAGA (SEQ ID NO: 10); and Reverse primer: CAATGCTGGTGCTGAATGAAA (SEQ ID NO: 11). According to a third embodiment of the invention, there is provided a primer pair for use in amplifying the PPP3CB gene, the primer pair comprising: Forward primer: CCTCATGCCCTGGAATACAA (SEQ ID NO: 7); and Reverse primer: CCAACTTAGACAGGAGACCATAC (SEQ ID NO: 8). According to a further embodiment of the invention, there is provided a primer pair for use in amplifying the TRPM2 gene, the primer pair being selected from the group consisting of: a) Forward primer: TCCGGCTCATGCACATTT (SEQ ID NO: 19); and Reverse primer: CAGCAGGAAGAGGAAGAAGAAG (SEQ ID NO: 20); b) Forward primer: ACAGCAACCACTCTCACTTC (SEQ ID NO: 22); and Reverse primer: CTGCTCCGATATGAACTTCTCC (SEQ ID NO: 23); c) Forward primer: CCTGTCTCGGACATCACTATCT (SEQ ID NO: 25); and Reverse primer: TTTGGTCCACTCGACAATCC (SEQ ID NO: 26); d) Forward primer: CTCTCTGGACTTCATCCTGTTC (SEQ ID NO: 28); and Reverse primer: TCCTTCATCATCCGCTTCAC (SEQ ID NO: 29); e) Forward primer: GGAGGTGTACAAAGGCTACAT (SEQ ID NO: 31); and Reverse primer: GTTCAGCTCCACGTCATTCT (SEQ ID NO: 32); f) Forward primer: CTTCTGGTTTGGTGTCAACAG (SEQ ID NO: 34); and Reverse primer: GCCTGAGATGGCAGATTTAATG (SEQ ID NO: 35); g) Forward primer: AAGAGGGCCTCCATGTTTC (SEQ ID NO: 37); and Reverse primer: CCCACGGAGTTTATCCTGAC (SEQ ID NO: 38); h) Forward primer: CCACTCTATGCGAACCACAA (SEQ ID NO: 40); and Reverse primer: CCAGGCTCAGGAGAGAGAA (SEQ ID NO: 41); i) Forward primer: CATCCACCATGGAGGTCATT (SEQ ID NO: 43); and Reverse primer: TTGAGGCACCTCGAAACAT (SEQ ID NO: 44); and j) Forward primer: AGGCGCATCCCACTCTAT (SEQ ID NO: 46); and Reverse primer: TGAGGGCACAGTCAGTAGT (SEQ ID NO: 47). According to a further embodiment of the invention, there is provided a kit for predicting COVID-19 infectiousness, the kit comprising: a) a primer pair for amplifying TBL1XR1; and b) a probe for detecting the TBL1XR1 gene. The kit may further comprise at least one primer pair and probe for amplifying and detecting PPP3CB, TRPM2 and / or MIR4323. The nucleotide sequences of the primer pair for amplifying TBL1XR1 may be: Forward primer: CCTCATGCCCTGGAATACAA (SEQ ID NO: 10); and Reverse primer: CCAACTTAGACAGGAGACCATAC (SEQ ID NO: 11). The nucleotide sequence of the probe for detecting the TBL1XR1 gene may be TTGCTTGGCTTCACCAGTATGTGC (SEQ ID NO: 12). The nucleotide sequences of the primer pair for amplifying PPP3CB may be: Forward primer: CCTCATGCCCTGGAATACAA (SEQ ID NO: 7); and Reverse primer: CCAACTTAGACAGGAGACCATAC (SEQ ID NO: 8). The nucleotide sequence of the probe for detecting the PPP3CB gene may be TCCAGGCAGCGGAACCATCTATTG (SEQ ID NO: 9). The nucleotide sequences of the primer pair for amplifying TRPM2 may be selected from the group consisting of: a) Forward primer: TCCGGCTCATGCACATTT (SEQ ID NO: 19); and Reverse primer: CAGCAGGAAGAGGAAGAAGAAG (SEQ ID NO: 20); b) Forward primer: ACAGCAACCACTCTCACTTC (SEQ ID NO: 22); and Reverse primer: CTGCTCCGATATGAACTTCTCC (SEQ ID NO: 23); c) Forward primer: CCTGTCTCGGACATCACTATCT (SEQ ID NO: 25); and Reverse primer: TTTGGTCCACTCGACAATCC (SEQ ID NO: 26); d) Forward primer: CTCTCTGGACTTCATCCTGTTC (SEQ ID NO: 28); and Reverse primer: TCCTTCATCATCCGCTTCAC (SEQ ID NO: 29); e) Forward primer: GGAGGTGTACAAAGGCTACAT (SEQ ID NO: 31); and Reverse primer: GTTCAGCTCCACGTCATTCT (SEQ ID NO: 32); f) Forward primer: CTTCTGGTTTGGTGTCAACAG (SEQ ID NO: 34); and Reverse primer: GCCTGAGATGGCAGATTTAATG (SEQ ID NO: 35); g) Forward primer: AAGAGGGCCTCCATGTTTC (SEQ ID NO: 37); and Reverse primer: CCCACGGAGTTTATCCTGAC (SEQ ID NO: 38); h) Forward primer: CCACTCTATGCGAACCACAA (SEQ ID NO: 40); and Reverse primer: CCAGGCTCAGGAGAGAGAA (SEQ ID NO: 41); i) Forward primer: CATCCACCATGGAGGTCATT (SEQ ID NO: 43); and Reverse primer: TTGAGGCACCTCGAAACAT (SEQ ID NO: 44); and j) Forward primer: AGGCGCATCCCACTCTAT (SEQ ID NO: 46); and Reverse primer: TGAGGGCACAGTCAGTAGT (SEQ ID NO: 47). The nucleotide sequence of the probe for detecting the TRPM2 gene may be selected from the group consisting of: k) TGTGAAGCGGATGATGAAGGACGT (SEQ ID NO: 21); I) TGGTCCTCAGAGGAATCTCCACC (SEQ ID NO: 24); m) TGATCCAGCAGAAACTGAGCGTGT (SEQ ID NO: 27); n) AAATGTGCATGAGCCGGAGGC (SEQ ID NO: 30); o) ATGACCCGAGGAACACGGACAATG (SEQ ID NO: 33); p) CTCACAGGAGCGTGAACCATGAGG (SEQ ID NO: 36); q) AGGTGCCTCAACATGGAGCCTT (SEQ ID NO: 39); r) CTGAGGGCACAGTCAGTAGTGAGC (SEQ ID NO: 42); s) CCCTCAGGCCTATGTCTGTGAGGA (SEQ ID NO: 45); and t) TTCTGGAGGAGGGTCTTGTGGTTC (SEQ ID NO: 48). The kit may further comprise one or both of the following internal control primer pairs: a) Forward primer: GCTACGCATCCCTGCTATTT (SEQ ID NO: 13); and Reverse primer: AGGTACTGGTCCTTATCCAGAG (SEQ ID NO: 14); and / or b) Forward primer: GTCACTTCGTGGCTAAGGTAAG (SEQ ID NO: 16); and Reverse primer: CTCAGCCCTGACACGTTAATAC (SEQ ID NO: 17). The kit may further comprise at least one probe having a sequence selected from: AGCGTGGACTTCAGTGCATTCATGA (SEQ ID NO: 15); and ACACAGACTCACCAAGCCACAGAC (SEQ ID NO: 18). According to a further embodiment of the invention, there is provided a method of predicting COVID-19 infectiousness and treating a subject who is infectious, the method comprising: determining expression of the TBL1XR1 gene in a biological sample from a subject having or possibly having COVID-19; optionally also detecting expression of at least one of the PPP3CB, TRPM2 and MIR4323 genes; wherein the level of expression of TBL1XR1 or at least one of the other genes indicates the likely infectiousness of the subject; and administering a therapeutically effective amount of a COVID-19 treatment to an infectious subject. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Study overview and scientific experiments. (A) Participants (n=59) were screened at COVID-19 testing facilities and the next day underwent evaluation using the cough aerosol sampling system (CASS) if the PCR and / or rapid antigen test was positive and within 7 days of symptom onset (visit 1; n=44). A follow-up visit was scheduled 48-72 hours thereafter (visit 2; n=38). (B) CASS was undertaken using a six-stage Anderson cascade impactor connected to cough tubing leading to a mouthpiece. A settle plate was included for collection of airborne SARS-CoV-2 in the cough cubicle (participant asked to count from 1-100 as loud as possible prior to cough sampling). Upper respiratory samples (nasopharyngeal swab, saliva) were also collected. (C) Venous blood to evaluate host transcriptomics and soluble biomarkers such as neutralizing antibodies were collected over a 48-hour period (visit 1 and 2). (D) &(E) All respiratory samples were assessed for SARS-CoV-2 by quantitative RT PCR and positive samples were further investigated for potential infectiousness by viral culture. Culture positivity was ascertained using a combination of cytopathic effect visualised using a light microscope (D) in tandem with confirmation of longitudinally increasing viral load determined by PCR (E). Representative micrographs are shown for baseline respiratory samples collected from participant CASS013, displaying cells at 24 h and 72 h post-infection. Samples showing the cytopathic effect (denoted by red arrow) have been highlighted by the bordered micrographs (clearing of the cellular monolayer). The viral supernatant was collected at 1,3, 6 and 9-days post-infection and tested by qRT PCR. Viral culture positivity was defined as an increase, of at least 100-fold, in viral copy number over the 9-day duration of culture. Viral culture experiments were duplicated for 10% of all samples to assess reproducibility of the technique. Figure 2: (A) Proportion of participants whose samples were culture-positive on non-aerosol (NP swab, cough tubing, saliva and settle plate) and aerosol (<10 pm and <5 pm) at visit 1 and visit 2. (B) Proportion of size-fractionated aerosol samples that were culture-positive at visit 1 and visit 2. (C) Proportion of participants with or without respiratory symptoms producing culture-positive samples (NP swab, cough tubing, saliva and settle plate) and aerosol (visits aggregated, i.e visit 1 (64) + visit 2 (18) = 82 visits in total). (D) Proportion of participants producing culture-positive samples and aerosol based on duration from symptom onset to sampling <8 days or >8 days (visits aggregated, i.e., visit 1 (73) + visit 2 (9) = 82 visits in total). Viral culture positivity was defined as an increase, of at least 100-fold, in viral copy number over the 9-day duration of culture. The Fisher’s exact test was used for comparisons between groups in (C) and (D). Figure 3: (A) Proportion of samples that were culture-positive in non-aerosol and aerosol (<10 pm and <5 pm) for the Beta, Delta or Omicron variants (visits aggregated, i.e., visit 1 + visit 2 = 82 visits in total). (B) Neutralization of SARS-CoV-2 pseudovirus by patients’ sera (n=18 for negative, n=7 for <5pm; n=7 for <10pm). Data are presented as median values +1- SEM. (C) Neutralization activity in aerosol culture positive and negative persons (mean of each group with standard deviation as error bars; n=18 for negative, n=7 for <5pm; n=7 for <10pm). (D) Nasopharyngeal Ct values in the aerosol culture negative (n=13) and positive participants (n=31); NP Ct values were used from visit 1 (except in 4 participants who were culture +ve on visit 2 but not visit 1). (E) Performance outcomes of nasopharyngeal Ct as a predictor for culture positive aerosol (sensitivity, specificity etc. expressed for various Ct cut-points incorporating rule-in, rule-out and Youden’s index readouts). The neutralization capacity (C) and nasopharyngeal Ct (D) are depicted by box-and-whisker plots indicating the median (middle line), 25th (bottom line) and 75th percentiles (top line), and the range (whiskers) of the measured parameters. Mann-Whitney test was used for comparisons across groups in (C) and (D). Figure 4: ROC for nasopharyngeal Ct as a proxy for identification of probably highly infectious cases. Figure 5: Selection of genes best predicting infectiousness as measured by capacity of patients to emit culture positive aerosols. PPV: Positive predicting value; NPV: Negative predictive value; AUC: Area under curve.; MIR4323: microRNA 4323; TBL1XR1: transducing (beta)-like 1X-linked WD40 repeat-containing gene, PPP3CB: protein phosphatase 3 catalytic subunit beta; TRPM2: transient receptor potential cation channel, subfamily M, member 2. An F-test was used to compare CASS-positive to CASS-negative participants. DETAILED DESCRIPTION OF THE INVENTION A method for predicting or determining whether a subject infected with SARS-CoV-2 is likely to be probably infectious or not is described herein. The method includes determining expression of at least one of the TBL1XR1 gene, the MIR4323 gene, the PPP3CB gene and the TRPM2 gene in a biological sample from a subject, wherein the level of expression of at least one of these genes indicates that the subject is likely to be infectious. The subject will typically have been diagnosed or confirmed as having COVID-19. As used herein, a patient is regarded as being infectious if they are capable of transmitting the SARS-CoV-2 virus, and in particular if they are capable of aerosolising culturable virus (i.e. emitting virus-positive aerosol particles). In various embodiments, the method comprises detecting the expression level(s) of: - TBL1XR1; - PPP3CB; - TRPM2; - MIR4323; - TBL1XR1 and PPP3CB; - TBL1XR1 and MIR4323; - TBL1XR1 and TRPM2; - TBL1XR1, PPP3CB and TRPM2; - TBL1XR1, MIR4323 and PPP3CB; or - TBL1XR1, MIR4323 and TRPM2. The biological sample is typically a blood sample, and may be whole blood, serum or plasma. The expression level of the one or more genes can be determined by determining the mRNA level of the genes in the sample, typically by first performing total RNA extraction, isolation of mRNA, reverse transcription to double stranded cDNA, clonal amplification of these cDNA, transcript assembly and finally RNAs differential expression analysis. A diagnosis of the subject being infectious for COVID-19 can be made when the level of at least one of the genes in the sample is higher (e.g. TBL1XR1, PPP3CB, TASOR, ZHX1, ARL8B, CWC22, TNPO1, CDC5L, CNOT8, SMC3, RIMOC1, KPNA3, ABRAXAS2) or lower (e.g. DNASE1L2, TRPM2, UBAP1L, VASN, ABCA3, BRSK2, SEMA6C, MIR4323, FTCD) than a typical level of the same gene in subjects who are not infectious. Cut-off (or threshold or reference) values can be determined based on typical levels of the same gene in subjects who have been diagnosed with COVID-19 but who are not infectious, in subjects who have been diagnosed with COVID-19 and are infectious, or in subjects who do not have COVID-19. The cut-off value can be a cycle threshold (Ct) value obtained from previous PCR reactions. In one embodiment, a positive diagnosis of infectiousness can be made when the expression of any one gene of interest in the sample is higher (or lower) than a reference level. In another embodiment, a positive diagnosis of infectiousness will require that expression of two genes in the sample be higher (or lower) than reference levels for those two genes. In another embodiment, a positive diagnosis of infectiousness will require that expression of three genes in the sample be higher (or lower) than reference levels for those three genes. Targeted interventions could be applied to individuals identified in this way as being likely to be infectious, such as to target or implement oral drug therapy, contact precautions, quarantine, improved ventilation, shielding of immune-vulnerable persons, and targeted isolation in workplace settings (e.g., workers in healthcare facilities and retirement homes, etc.). Drug therapy could include administration of a therapeutically effective amount of a treatment for COVID-19, in particular a treatment which reduces the risk of onward infection. These include antiretrovirals, such as nirmatrelvir / ritonavir (e.g. Paxlovid™ by Pfizer) or remdisivir. Conversely, a subject who is not identified as likely to be infectious could avoid any of the above precautionary steps. In order to determine the target gene(s) in the sample by PCR, primers which are specific for a target region of each gene are required. By primer, it is intended to designate short nucleic acids, generally DNA oligonucleotides 10 nucleotides or more in length (such as 10-60, 15-50 or 20-30 nucleotides in length). Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence. A target region for each gene can be identified by methods known in the art and software programs are available for designing primers which can hybridise to and amplify the target region. Probes which designate a complementary nucleic sequence of about 10 or more nucleotides long and to which detectable molecules or label are attached such as for example fluorophore or fluorescent label can also be designed. "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self 17 hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the "complement" of the given sequence. As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part 1, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y. "Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. The detectable labels on the probes can be fluorophores, fluorescein, etc. Each probe will typically have a different label. The exon sequences for the target genes identified herein (TBL1XR1, PPP3CB, TRPM2 and MIR4323) as well as for the housekeeping genes Ring finger protein 213 (RNF213) and Actin gamma 1 (ACTG1) are provided in Table 1. Table 1: Complete exon sequences of target genes Protein phosphatase 3 catalytic subunit beta [PPP3CB] (SEQ ID NO: 1) Homo sapiens protein phosphatase 3 catalytic subunit beta (PPP3CB), transcript variant 2, mRNA >ref|NM_021132.4|:1-3519 ATGCCGGTCTTAGCCCCTCTGTAGGGAAAGAGGGTCCGCCATGTTCCCCGGCGGCGCCGCCGCTTGGCTC TGGTAGCCGCCGCCCCCGCCCCCAACCCCGCCCGGCCCAGAGCCTAGCCGAGCCCCGGGCCCAGCATGGC CGCCCCGGAGCCGGCCCGGGCTGCACCGCCCCCACCCCCGCCCCCGCCGCCCCCTCCCGGGGCTGACCGC GTCGTCAAAGCTGTCCCTTTCCCCCCAACACATCGCTTGACATCTGAAGAAGTATTTGATTTGGATGGGA TACCCAGGGTTGATGTTCTGAAGAACCACTTGGTGAAAGAAGGTCGAGTAGATGAAGAAATTGCGCTTAG AATTATCAATGAGGGTGCTGCCATCCTTCGGAGAGAGAAAACCATGATAGAAGTAGAAGCTCCAATCACA GTGTGTGGTGACATCCATGGCCAATTTTTTGATCTGATGAAACTTTTTGAAGTAGGAGGATCACCTGCTA ATACACGATACCTTTTTCTTGGCGATTATGTGGACAGAGGTTATTTTAGTATAGAGTGTGTCTTATATTT ATGGGTTCTGAAGATTCTATACCCAAGCACATTATTTCTTCTGAGAGGCAACCATGAATGCAGACACCTT ACTGAATATTTTACCTTTAAGCAGGAATGTAAAATTAAGTATTCGGAAAGAGTCTATGAAGCTTGTATGG AAGCTTTTGATAGTTTGCCTCTTGCTGCACTTTTAAACCAACAGTTTCTTTGTGTTCATGGTGGACTTTC ACCAGAAATACACACACTGGATGATATTAGGAGATTAGATAGATTCAAAGAGCCACCTGCATTTGGACCA ATGTGTGACTTGTTATGGTCCGATCCTTCTGAAGATTTTGGAAATGAAAAATCACAGGAACATTTTAGTC ACAATACAGT T C GAGGAT GT T CT TAT T T T TATAACTAT C CAGCAGT GT GT GAATT T T T GCAAAACAATAA TTTGTTATCGATTATTAGAGCTCATGAAGCTCAAGATGCAGGCTATAGAATGTACAGAAAAAGTCAAACT ACAGGGTTCCCTTCATTAATAACAATTTTTTCGGCACCTAATTACTTAGATGTCTACAATAATAAAGCTG CTGTATTAAAGTATGAAAATAATGTGATGAATATTCGACAGTTTAACTGTTCTCCACATCCTTACTGGTT GCCTAATTTTATGGATGTCTTCACGTGGTCTTTACCGTTTGTTGGAGAAAAAGTGACAGAAATGTTGGTA AATGTTCTGAGTATTTGCTCTGATGATGAACTAATGACTGAAGGTGAAGACCAGTTTGATGGTTCAGCTG CAGCCCGGAAAGAAATCATAAGAAACAAAATTCGAGCAATTGGCAAGATGGCAAGAGTCTTCTCTGTTCT CAGGGAGGAGAGTGAAAGTGTGCTGACACTCAAGGGCCTGACTCCCACAGGGATGTTGCCTAGTGGAGTG TTAGCTGGAGGACGGCAGACCCTGCAAAGTGCCACAGTTGAGGCTATTGAGGCTGAAAAAGCAATACGAG GATTCTCTCCACCACATAGAATCTGCAGTTTTGAAGAGGCAAAGGGTTTGGATAGGATCAATGAGAGAAT GCCACCTCGGAAAGATGCTGTACAGCAAGATGGTTTCAATTCTCTGAACACCGCACATGCCACTGAGAAC CACGGGACGGGCAACCATACTGCCCAGTGACCCACTACTTCCCAGGGACTCTCACATCTCGGGCCCCAAA TGGACAGATCACCCGAGGAGCTGGAGGGGTCGGCCAAGCTGACTGTAAATTTCACAGTCTCTCTGAAGAA ACCATTGTGCTTCTGAGACCCTAGCCCCCTTCCTGGATGGAGGCTTGAGGGCCCTGGGACATGTGCTATC TGATAAGATTGGGTCATCGCTGCCAAGGTGGAGAGCAGTGAGCAAGGGGCTTGGGGCAATTTCCAGTGGA GGGCATCCACACCTCCATTTTATGCTTGTGGTTCACACATTTAAGTTTACAAATCAGATTTCTTTTCCCC TTCAGTAGAATTAGATTTTGTTTTTCAATCATGATTTCAAATGCAATCCTAAGAGCTAATGTGGACTTTT CTTTTTCCATGAAATGTCTTTAAAGGATGAATTAGCATGGTCTTAAAATACATTTCTGAGGTTACTAGCT GTATTTTGAATTGTGAGCAAAATGCCGAGAAACCCAGTTGGCATTTATACAAAATGTTGACCTCAGGTCT ATAGTTCTTAAATGTGGCTAATTCTGTAACATAGTCTTGGTATTTTTTAATTATGAATGCATATCCTATT TCCAGGCAGGCTCTCTTACTTGAACACAAATCCAAAAACTAATTTAGAGTCTTTTTTGCCCAGATCTTTT AAGACTTACACCCCAGAGATTTAAGAAGAAAACCTCTAAATTTCAAAATTATGAAGAATTACAGAATTAC TCATTTAAGGTACTTTAAAAGAAGTTTGTACATTGTCAAAGTAAATTTTAATTCAAATCATGTCTGTAAA ACTTGACGTATTTTGTGTATGCATGTTTTCATTTTGCAAATATTTAATATATAGACCTATGATGTACAGG TACGACATGTATAGGTTACCTAGATGTTATGAGAAATTTTAGTTTATTGTGAGTACTCAAGTTGCTTAGA GAGCCACCAGGGTGATTTGCTGCTGGCTTTCTATCATTTTTATGTTTTAATGCAAAGGAAATTTTAAAAT GTTCTGGAAGTGTTTTTGATTAAGCAATGCAGCCTAGAAGCAATGGTTCTGTTCAATCATTCAGATGTTA GTGGAAGCATAAAAGTCAAGACTGCATGTTGAAACCTTTCTTTTGATAGTTACTGAACTGCTTGGTTAAA CTAAATGGAACCATGTGCTAATTTTTCACAATTATTGACCTGTATTGATTGCCACTGTAGTTTGGTATTT CCCTTTACTTTGGTGGCCTGCTTCCCTCATGCCCTGGAATACAACTCAGAGCTCCAGGCAGCGGAACCAT CTATTGTTTTGTTTGCCAGAAAGTGCACCCTGTATGGTCTCCTGTCTAAGTTGGAAATATTATGCATGTG CAGGACTATTCGAGTATTTTATAAACAGTAGCACACAATAAATTCCATGCATGGGCCGCTGCTCCTATCT CTGTGTTGGGTTTTATTTGGAAGATGCAATCTGATTTGTCCTTTTGATGCAAATCAGAAAATCCTGTTAC TAGAGCTGGGATGTCCTCCGGAGATTATCTCGTGGATAGTTCATGGTAATTTGATTAATTAAATTCTTTA TAAATTTTGCCTTAAAAAAAACTTTTGTTATATACTTGTTTTACATGAGCATTAGTAACTGAGCACTAGA GGACTTTGAATGCCTACTGTAGGCCTCCTAAGTCTAATATTTAAGATCACTGTTTATTGTCTTTTAATTG AAAGAAAATATGTTATTGTCTAGAATTTTGTTATAGTGGTATTGGGAATTTACTGGGTGTTCTAACAATA AGAAAAATAT T AGT GAT AA TBL1X receptor 1 [TBL1XR1] (SEQ ID NO: 2) Homo sapiens TBL1X / Y related 1 (TBL1XR1), transcript variant 1, mRNA >ref|NM_024665.7|:1-8182 GAACCGTTTGGCTCTTTCCCTTAGTAACAGCTTTTACCTTCTGCCTCAATTCCTCTCCCCGCTCGCGCCC TTCCCCCGCGCGCCGCCTCCCCCCGCCGCCGGCCGCGTGCGCCCCCGCCCCTCCCCCCGCGGCGGGAGCA CCTCCGCTCGCCACCTGGGGACCCGCGCCGCGCTCCCCGCCCCCGCCCGCCCGGCGCCCATCCCCCGCGC CCTCCGCTCGCTGGGGTTATAATTGCCTCTCACCCCCCGGAGGGGTTATTTTGGGGGTGGTTGGAGGCGG TGGCGGCGGCGGCGAGGAGGGGAATTTCCTTGTGCCTCCATTCCCGGGAGGGGGGAGCGGCGTTGGAGGC CACCGTTTCCAGCATCAACAACAGCAACTTGTGATTGGCGGTGACCGGATATTCAGTTGCACATCCCCAC ATCAATGCACTGCCAATGGGTTATATCCTGTGTTGTGACCTCATGGTTTAAGTGGGAATAAAGATGAGTA TAAGCAGTGATGAGGTCAACTTCTTGGTATATAGATACTTGCAAGAGTCAGGATTTTCTCATTCAGCATT TACCTTTGGTATAGAAAGCCATATCAGTCAGTCCAATATAAATGGTGCCCTCGTCCCACCCGCTGCATTG ATTTCTATCATCCAGAAAGGTCTACAGTATGTAGAAGCAGAAGTTAGTATTAATGAGGATGGTACCTTGT TTGATGGTCGACCAATAGAGTCTCTGTCCCTGATAGATGCCGTAATGCCTGATGTAGTACAAACAAGACA ACAAGCTTATAGAGATAAGCTTGCACAGCAACAGGCAGCAGCTGCTGCAGCTGCCGCAGCTGCAGCCAGC CAACAAGGATCTGCAAAAAATGGAGAAAACACAGCAAATGGGGAGGAGAATGGAGCACATACTATAGCAA ATAATCATACTGATATGATGGAAGTGGATGGGGATGTTGAAATCCCTCCTAATAAAGCTGTTGTGTTGCG GGGCCATGAATCTGAAGTTTTTATCTGTGCCTGGAACCCTGTTAGTGATCTCCTAGCATCAGGGTCTGGA GACTCAACAGCAAGAATATGGAATCTTAGTGAGAACAGCACCAGTGGCTCTACACAGTTAGTACTTAGAC ATTGTATACGAGAAGGAGGGCAAGATGTTCCAAGCAACAAGGATGTCACATCTCTAGATTGGAATAGTGA AGGTACACTTCTAGCAACTGGTTCCTATGATGGGTTTGCCAGAATATGGACTAAAGATGGTAACCTTGCT AGCACCTTAGGGCAGCATAAAGGCCCTATATTTGCATTAAAATGGAATAAGAAAGGAAATTTCATCCTAA GTGCTGGAGTAGACAAGACTACAATTATTTGGGACGCACATACTGGTGAAGCCAAGCAACAGTTTCCTTT TCATTCAGCACCAGCATTGGATGTTGATTGGCAGAGCAACAACACCTTTGCTTCTTGTAGTACAGATATG T GCAT T CAT GT CT GTAAAT TAGGACAAGACAGACCTAT TAAAACAT T C CAAGGACATAC GAAT GAAGTAA ATGCTATCAAATGGGACCCAACTGGCAATCTCTTGGCCTCCTGTTCTGACGACATGACTTTAAAGATATG GAGTATGAAACAAGACAATTGTGTCCATGATTTGCAAGCACATAATAAAGAAATTTATACTATCAAATGG AGTCCAACAGGACCAGGGACTAATAATCCAAATGCCAACCTTATGTTAGCAAGTGCATCCTTTGATTCTA CTGTTAGGTTATGGGATGTAGACCGAGGGATATGCATCCATACCTTGACAAAACACCAAGAGCCTGTGTA CAGTGTAGCTTTCAGTCCTGATGGCAGGTATCTGGCAAGTGGTTCTTTTGACAAATGTGTACACATCTGG AACACGCAGACAGGTGCTCTAGTTCACAGCTATAGGGGAACAGGTGGAATATTTGAAGTTTGCTGGAATG CAGCAGGAGACAAAGTTGGAGCCAGTGCATCAGATGGTTCAGTTTGTGTATTAGACCTTCGGAAATAGCG CTACTAGTTGGAAGCCATGGACCGACTATGAATGTGTACATAGCCAAAATGACTGTCCCTGACCCATGTA CTGCTATAGTCCCACTTGAACCATGGCCAGTCCACTACAGCCAAATCTAAAAGAAATATATACATACAGT GTATATAAACAAAATTGCACCCTGAAGATGACAGAGTTTTGTCACAGCTTGTGAATTCTGTTCACCAAGT GCTGGAATCTAATCTGCTGTGCCCCTAAAATAGCATTTAGAAGTTTTGGATATGAAAAACAGAAGAGAGA AAAATATACATTATAAAAGCAGAACATACATGTACCAGTTTTTGGATACTAAATGACAGCCTTGTTTCTC CCCTTTGAATCAGCAGACACCATGGATTATATTCTTTTTTTCCCTTCAGTAGTGAGCAGTTTGTATGTAC AGAGAAAATGGACTTACAAAAACTTGCAGCAGTAGTTTGTTCTTGCTTTAAAATTTCGTTTTTGGTTTAG ATTATGGATGCATGAAGTAAGGGAGTGAATCAGTTTCTTGTTTATATTTTTTTCACCTTTTAAACAAAAA ATTCTTTAAAATATTTTAATGCATTCTTTTGAAGAGGTAGATGTTTGGTACATTTTATGGCTCCCAGAGC ATATATTCAGTTGGTGCATGTTGTGGAAGGGGGAATTGGAAATTAAATGAAAACCTATGACTTTGGTCAT GTCAATCTGTAAGACACATCAGTAAAAGGGTATTATGCTCTGTTGGTTTTGTTTTTTTGTTTTGCTTTTT TTTTTTTTTTTTCTTTTTTGTTTTTTGGTGATGTGGCTTaaaTgcaaTAGTTTCTTTTTTgggacaTATT TCTGCCAATTAAAGACTAGAAGGGCACAACTTTTTTTTTAATTACCATAGAGAAGATACATTAAAAAAAA TCTTCTGATGTTTTGTAGCCATAACTAAATTATGGTAAAAATGTGCACTATTGTGAAAAGGAGCAACGTA GTTTTGGGTTTTTTGTTGTTTGTTTGTTTTGCTTTGTTTTTTAAGAGATTAAAATGTTTCTGGATAAGGA TTAGCTTCTCGAAGTGTCCATCATTCTGTGTAGAAGCTTAAATATGTAATGTAACCAAACTCCAGTATTA AAAATCTCTCATGTTGTTTTCTTTATACAAAGCAAGATAACGGCATATAACACTGCCATTACATGGCAAA ATGTTTGCTACCTTAGTTTAAAAAACAATCTCAAACAAAAGACTTGCTTCAAGGTGTTTTTAAATAGCAG TGATTCAGAATTTTTTTTAATGAAAGTATAATTGCACTAACCTTCTTCCTGCTGCTCTGATTCTGCATTT GTGGTACTTGTGACTACGTTTTTTCAAATATAGATAGATTTAAGCTGCTAATTTTTTTTTTTTTAGTAAT CACTACTATATCATGTCTTTTACTCTGTTTATAATATCAAGTATTTTCTTAAAGATATAGATATTAAACC TTGTGCTCATGCAACTTAGAGTAACATATACAGACAAATGATTGCATGAGGCCATGTTTATATGTGTGAC T AAT AAG G C T T GT CAT GAT T AACAT AAT C CAGGT AT GT CAT T T C T GAAGAGAATAGT CAT CAAAT T TATA TCTCGAAGATTTTAATTAAGGAATTGCTTATTGTTGAGCTTAGCAAATTAATAACACTATTTCTGTCACT AATTATTTTGAGGCCTTTTAGTACTAAAATTTTAACCTGTGTTCTAAGTAGAAACTGATTTAACCCAAGT AATGCAGCTTTGATTGATTTCAGCATTCGTTGCTTTGCTATTTTTACAAAACAGCATTGATTGAAGCAAG TCTTGGTTTTACTAAGGTAGGGTAGCATTTGCTATTGGTAAAGAGAATAAATACACTTAATTTCACAATA CATTGTTATATGTACCCCAGTTGTTGTTAGTGGGGACTATGATACTGTAATAATATTTTTAAAAATTTAC ATCAAGAGAGGCAGTCATTCACGATGGTTTTGTGCCAGCTCTTTTTAGGGTTTTGGATCACATTAGAGAT ATTTAGAACATATTACCCTGTGACTTACGTAGGAAACCTAATATGCTGAGTATCTGGCACTTGAATTCCT GCTTTTATTGCTGGAGGTCCACATCTGTGGTTGACCTCTGTTATTGTTTAAAAAAAATAAATAAAAATTA AAAAAATCTGTGCAATAATTTTAAAATGTGCTCCCAGGAATAGACACAAATGTTTTGAGTATCTTTTAAG CTGCATTTTCCTTTAGTGATGCATTTGTCAATTGCACTGAATTTAAATCTGAAAGTCAGAGGTGATTATT GATAGTACTTTTGTATTTTGATATGGACAGTTTATTCATTTGCATACAGTTATTGACTTTTTCCCAGCTG AT TAAAAGATAGT CAAGAAAT T CT GCAATATAGCT GC CAAAATAGACAGCTACAT T T T TAT GATAT T GT C ATCTTTTCTGTTTTTTTTTTCTTTTTTTTCTTTAGCTATTTTACTTAAGCATAATAGCCACAATAGGACA TATAAAAGATTATAAATACAGAGCTTTATTATCCTGACGTCTTGGGTCTTTTAAGTATATACTTTTCTGA AAGGTATCCATTTTGTAGGCTTGGGTTCTTCATGAGCATACGATTGTTTATTTTTGCTGCTGTTCTCAAC ATCATCATTGCCTGCTGATGTGCCACGATGCTGCTCCAATAGACAGCAATAAGATTGTCTCTAATTTGAG CAGTAACATGATTGCAAGAGACCAAGTTTCACAGCTTGTAAAGTTCTGTATTTGGGATTCTTGCTTATTT TTCCGCCTGTGTTTTTCTGAGAACTTATTCCTGATGATCAATTGAATCCAGTAGTTTTTCTATGCTATTT GT T GT T GT AT AAGCTACT GT AAGAAACT T ATCATAAGGAAAAAT AGAAAGGAAAACT T GAAT CAAT ACT C ATTGATTAAAATGGAATAAAGAAAGAGCAGCTGCCACTTTTAAACAACATAAAGGAATATCTTTTTTTGT CTCCGTGTAGGAAATCCCATAAGTTCTTATATTTGTTCCAGTTCCCATTTCCTGCCATTGACCAGATAAC ATCATTGACTTTCAAATGACTTTTAGAAGTGATAACTCTTAATTTCCTAATAGATACTAGATTGTATTGA ATTCTGTTTTAATTATTCTCTAGGTAAGTATGTTTTAGGATTAAATACCTTTTACAGATACTGAAAGTGC CTCCTTTTGTGGTGTAAAAAACAAATTATGGTGCAAAAAGTAATCACTAGATTGAAATACATGAAGGTTT TTTGCTTTTTGACATACGAAAATGTCAAGAGAAAGGCCAAAGATTTGTACTTTTTCACTTACAAAGCACT CCTTTTTCCCTTTWkCTTCTTTCTGTC^AATTAGATTTTW^TGAGAGAGTACTATTTTTAAGGAGCTATCT GTTTATGTAGAATGATTTTGTTAAGAGTAATGTAAACTATTATTGAGTAGAGGCCTAAAGAGGACTGTGC ATTTTTGCTATTTAAAGGAATCACAAATGATCATACTTAAGTGAGGAAAAATGACAAGTTTTACTAGCTA AGTAGAGAAATAAATCTCAAATGCAGCGCTACAATTTTCATTATCTTAAGTACATTGTACATTTCTACAG AACCTGTGATTATTCTCGCATGATAAGGATGGTACTTGCATATGGTGAATTACTACTGTTGACAGTTTCC GCAGAAATCCTATTTCAGTGGACCAACATTGTGGCATGGCAGCAAATGCCAACATTTTGTGGAATAGCAG CAAATCTACAAGAGACCCTGGTTGGTTTTTCGTTTTGTTTTCTTTGTTTTTTCCCCCTTCTCCTGAATCA GCAGGGATGGAAGGAGGGTAGGGAAGTTATGAATTACTCCTTCCAGTAGTAGCTCTGAAGTGTCACATTT AATATCAGTTTTTTTTAAACATGATTCTAGTTAAATGTAGAAGAGAGAAGAAAGAGGAAGTGTTCACTTT TTTAATACACTGATTTAGAAATTTGATGTCTTATATCAGTAGTTCTGAGGTATTGATAGCTTGCTTTATT TCTGCCTTTACGTTGACAGTGTTGAAGCAGGGTGAATAACTAGGGCATATATTTTTTTTTTTTTTGTAAG CTGTTTCATGATGTTTTCTTTGGAATTTCCGGATAAGTTCAGGAAAACATTCTGCATGTTGTATCTAGTC TGATGTACTTATCCATCTCATTACAAACAAAAACACACAGACTGCATTTGTAGCTCTGTAATCCTTGAAT ACGGAAGTAAATTTTCTTCTTTCCTGACTTTGACATTGTAGCTATACTGTTTCCATTTTTGTTTTTACAA ATCCTTTGGGTCTAATTCTGTGAGCCTACCTATAGCACTGGATTAAAATGTCTGCATCATTTCTTTAGTT ATCCAGTTAACTTTAAAACTGTTGTAAAAGTGTAAACCAGCCCATGACAGGTTTTTGTACATGTTAAAGA ACTTCATTGTTCAGTTTTCATGATTATTGTGTAAGGAAGACTGATGTAGATGTTCTGTGCTGTCCTGGAC CATGTTAATTACACTTACGACGTATTTTAGTTCCACATCACAATGATTTGTCCCCAGTGACCCTTTTATC CTTTCTAGGCACATTTCTTGTTGTTGTTGTTGTTGCAGTTCCCCTTTGCATTGTATTGCTTTGACAACTG TAATTTGAATCAGATCTGAAAGAGGTCCAGAATAAAATATATTTTGATATTATGTTGGCTGTGTACATAT ATAAAACCTTTGATGTCTATGTAGTTTATATAGACTATTTACTAGTCAGGTAAAGAGAGAGGGATGGTAT TTCTTGTTTACAGTTTGTTTACATTTTAATTGCTTTTACTTCTCCAGTGTTCTATTGTAGAATAAACCTT TTTTTCCAAGTTAGCCTTCATAACCTATAACACATTCATTCTCTCCCATACTGGTGTTACTGATTAATAA CTGCCATCAGAACCCAAACCACTGTTTTTCATAAACGTATTGTTTCCCAAGATATATACTTTAAGCCCAT AATAAAATTACAAATTTTACTATTTTTATTACTTCTTGCAGGAGATAAGATTTTTTTAAATGAAGGGGTT ATTTACTATTTTTATTAAAGTATAATAGAGCATCTAATCAGCTAAGGGATTGTAATTTTTAATTCTTTTG AAAAATAAATATTGTATTTAAAAGACGTTATTTCACAGAAGCTGAAAAAGAGACCTTAGATAACATTTGT TTGGTTAGCCACACGGTTGAGCACAAAACAATGTGTAGATGTGTTGAAGATTAGGGCAGGAGGCTCAACT TCTCGGTGACCTTTTTTTGCTTCACAACAAGCCAATTATAGTTGAATCATTTTCTCTCTTAGCTAGTTGT TACTACAAACTTTATAAGAAAAACAACTAGACACCTTCTAGTTTTAATTAATACCAACTCCTTTAGAGTT AGAGACTTTTTAAAAAGAATCATTGAGCATATTTTCTTTTTTTTTTTTTTAAGAATTTACACTCTCTAGG TCTTCTATTTTTCCTGTTTATTTCCTTAAACAGAAATAAATTCAAGCATAGTTTTATGTTTTATTAGAAA TAAGCCACTGCAGTTGCTAGAACTGACATGCTTTCATTTTCAAGGGGTTAACTTTTATTGCATGGCATAG ATTATTAGTAATATCCTGCATTGTATACTTTTCCAAAAAGGGTTGGACTCCTAAGGATTAGAATCCTTAT AAGCTTGAGGGAAAGCTGAAACTAATTTGAACAGTAATAGAAGGGATTATTTTTAATTTCATAGTTAACA CTTTACAATGGATTAGATTTATTACCTAGTATAATCCGTAGCAGATCAGATCAAAGTTTACTGTTTTACC AATATATTCATTTGAAAAAATACAGTGGAGGAAAAGACACTTTAGATGGTAGTTTTATGGTGCTTTTCTC TGCTGTACCAAAGTGCTTTGTCATCAATGAAATGTTAACCCTATGCTACTTTTTCTGCATGCTTCTTAAA TGTCATCTTAATGCTTAATTGTCCTAACTTGTAATATTTTTGTATTAAATTTTTTTAGGAGTTCCTTGTA AT T T C T T TACAAT T TAAAATATAT TAAACACAAAGT GT T TAGTAAAGTACAGAAGACAAT TA Transient receptor potential cation channel subfamily M member 2 [TRPM2] (SEQ ID NO: 3) Homo sapiens transient receptor potential cation channel subfamily M member 2, transcript variant 1, mRNA >ref|NM_003307.4|:1-5419 AGAACCCCAGTGTAGCGAGCTGGAGAGAGGACTGTCCTGAGGGCAGCAGGCCTGGTTGCAGCTGGCGTGG GGGTCTCAGAATGGAGCCCTCAGCCCTGAGGAAAGCTGGCTCGGAGCAGGAGGAGGGCTTTGAGGGGCTG CCCAGAAGGGTCACTGACCTGGGGATGGTCTCCAATCTCCGGCGCAGCAACAGCAGCCTCTTCAAGAGCT GGAGGCTACAGTGCCCCTTCGGCAACAATGACAAGCAAGAAAGCCTCAGTTCGTGGATTCCTGAAAACAT AGACAGCAACCACTCTCACTTCATCCTCGTGGACGACGGGACCCACGGCCAGTACGGGGTGGAGATTCCT CTGAGGACCAGGCTGGAGAAGTTCATATCGGAGCAGACCAAGGAAAGAGGAGGTGTGGCCATCAAGATCC CCATCGTGTGCGTGGTGCTGGAGGGCGGCCCGGGCACGTTGCACACCATCGACAACGCCACCACCAACGG CACCCCCTGTGTGGTTGTGGAGGGCTCGGGCCGCGTGGCCGACGTCATTGCCCAGGTGGCCAACCTGCCT GTCTCGGACATCACTATCTCCCTGATCCAGCAGAAACTGAGCGTGTTCTTCCAGGAGATGTTTGAGACCT TCACGGAAAGCAGGATTGTCGAGTGGACCAAAAAGATCCAAGATATCGTCCGGAGGCGGCAGCTGCTGAC TGTCTTCCGGGAAGGCAAGGATGGTCAGCAGGACGTGGATGTGGCCATCTTGCAGGCCTTGCTGAAAGCC TCACGGAGCCAAGACCACTTTGGCCACGAGAACTGGGACCACCAGCTGAAACTGGCAGTGGCATGGAATC GCGTGGACATTGCCCGCAGTGAGATCTTCATGGATGAGTGGCAGTGGAAGCCTTCAGATCTGCACCCCAC GATGACAGCTGCACTCATCTCCAACAAGCCTGAGTTTGTGAAGCTCTTCCTGGAGAACGGGGTGCAGCTG AAGGAGTTTGTCACCTGGGACACCTTGCTCTACCTGTACGAGAACCTGGACCCCTCCTGCCTGTTCCACA GCAAGCTGCAGAAGGTGCTGGTGGAGGATCCCGAGCGCCCGGCTTGCGCGCCCGCGGCGCCCCGCCTGCA GATGCACCACGTGGCCCAGGTGCTGCGGGAGCTGCTGGGGGACTTCACGCAGCCGCTTTATCCCCGGCCC CGGCACAACGACCGGCTGCGGCTCCTGCTGCCCGTTCCCCACGTCAAGCTCAACGTGCAGGGAGTGAGCC TCCGGTCCCTCTACAAGCGTTCCTCAGGCCATGTGACCTTCACCATGGACCCCATCCGTGACCTTCTCAT TTGGGCCATTGTCCAGAACCGTCGGGAGCTGGCAGGAATCATCTGGGCTCAGAGCCAGGACTGCATCGCA GCGGCCTTGGCCTGCAGCAAGATCCTGAAGGAACTGTCCAAGGAGGAGGAGGACACGGACAGCTCGGAGG AGATGCTGGCGCTGGCGGAGGAGTATGAGCACAGAGCCATCGGGGTCTTCACCGAGTGCTACCGGAAGGA CGAAGAGAGAGCCCAGAAACTGCTCACCCGCGTGTCCGAGGCCTGGGGGAAGACCACCTGCCTGCAGCTC GCCCTGGAGGCCAAGGACATGAAGTTTGTGTCTCACGGGGGCATCCAGGCCTTCCTGACCAAGGTGTGGT GGGGCCAGCTCTCCGTGGACAATGGGCTGTGGCGTGTGACCCTGTGCATGCTGGCCTTCCCGCTGCTCCT CACCGGCCTCATCTCCTTCAGGGAGAAGAGGCTGCAGGATGTGGGCACCCCCGCGGCCCGCGCCCGTGCC TTCTTCACCGCACCCGTGGTGGTCTTCCACCTGAACATCCTCTCCTACTTCGCCTTCCTCTGCCTGTTCG CCTACGTGCTCATGGTGGACTTCCAGCCTGTGCCCTCCTGGTGCGAGTGTGCCATCTACCTCTGGCTCTT CTCCTTGGTGTGCGAGGAGATGCGGCAGCTCTTCTATGACCCTGACGAGTGCGGGCTGATGAAGAAGGCA GCCTTGTACTTCAGTGACTTCTGGAATAAGCTGGACGTCGGCGCAATCTTGCTCTTCGTGGCAGGGCTGA CCTGCAGGCTCATCCCGGCGACGCTGTACCCCGGGCGCGTCATCCTCTCTCTGGACTTCATCCTGTTCTG CCTCCGGCTCATGCACATTTTTACCATCAGTAAGACGCTGGGGCCCAAGATCATCATTGTGAAGCGGATG ATGAAGGACGTCTTCTTCTTCCTCTTCCTGCTGGCTGTGTGGGTGGTGTCCTTCGGGGTGGCCAAGCAGG CCATCCTCATCCACAACGAGCGCCGGGTGGACTGGCTGTTCCGAGGGGCCGTCTACCACTCCTACCTCAC CATCTTCGGGCAGATCCCGGGCTACATCGACGGTGTGAACTTCAACCCGGAGCACTGCAGCCCCAATGGC ACCGACCCCTACAAGCCTAAGTGCCCCGAGAGCGACGCGACGCAGCAGAGGCCGGCCTTCCCTGAGTGGC TGACGGTCCTCCTACTCTGCCTCTACCTGCTCTTCACCAACATCCTGCTGCTCAACCTCCTCATCGCCAT GTTCAACTACACCTTCCAGCAGGTGCAGGAGCACACGGACCAGATTTGGAAGTTCCAGCGCCATGACCTG ATCGAGGAGTACCACGGCCGCCCCGCCGCGCCGCCCCCCTTCATCCTCCTCAGCCACCTGCAGCTCTTCA TCAAGAGGGTGGTCCTGAAGACTCCGGCCAAGAGGCACAAGCAGCTCAAGAACAAGCTGGAGAAGAACGA GGAGGCGGCCCTGCTATCCTGGGAGATCTACCTGAAGGAGAACTACCTCCAGAACCGACAGTTCCAGCAA AAGCAGCGGCCCGAGCAGAAGATCGAGGACATCAGCAATAAGGTTGACGCCATGGTGGACCTGCTGGACC TGGACCCACTGAAGAGGTCGGGCTCCATGGAGCAGAGGTTGGCCTCCCTGGAGGAGCAGGTGGCCCAGAC AGCCCAAGCCCTGCACTGGATCGTGAGGACGCTGCGGGCCAGCGGCTTCAGCTCGGAGGCGGACGTCCCC ACTCTGGCCTCCCAGAAGGCCGCGGAGGAGCCGGATGCTGAGCCGGGAGGCAGGAAGAAGACGGAGGAGC CGGGCGACAGCTACCACGTGAATGCCCGGCACCTCCTCTACCCCAACTGCCCTGTCACGCGCTTCCCCGT GCCCAACGAGAAGGTGCCCTGGGAGACGGAGTTCCTGATCTATGACCCACCCTTTTACACGGCAGAGAGG AAGGACGCGGCCGCCATGGACCCCATGGGAGACACCCTGGAGCCACTGTCCACGATCCAGTACAACGTGG TGGATGGCCTGAGGGACCGCCGGAGCTTCCACGGGCCGTACACAGTGCAGGCCGGGTTGCCCCTGAACCC CATGGGCCGCACAGGACTGCGTGGGCGCGGGAGCCTCAGCTGCTTCGGACCCAACCACACGCTGTACCCC ATGGTCACGCGGTGGAGGCGGAACGAGGATGGAGCCATCTGCAGGAAGAGCATAAAGAAGATGCTGGAAG TGCTGGTGGTGAAGCTCCCTCTCTCCGAGCACTGGGCCCTGCCTGGGGGCTCCCGGGAGCCAGGGGAGAT GCTACCTCGGAAGCTGAAGCGGATCCTCCGGCAGGAGCACTGGCCGTCTTTTGAAAACTTGCTGAAGTGC GGCATGGAGGTGTACAAAGGCTACATGGATGACCCGAGGAACACGGACAATGCCTGGATCGAGACGGTGG CCGTCAGCGTCCACTTCCAGGACCAGAATGACGTGGAGCTGAACAGGCTGAACTCTAACCTGCACGCCTG CGACTCGGGGGCCTCCATCCGATGGCAGGTGGTGGACAGGCGCATCCCACTCTATGCGAACCACAAGACC CTCCTCCAGAAGGCAGCCGCTGAGTTCGGGGCTCACTACTGACTGTGCCCTCAGGCTGGGCGGCTCCAGT CCATAGACGTTCCCCCCAGAAACCAGGGCTTCTCTCTCCTGAGCCTGGCCAGGACTCAGGCTGTTCCTGG GCCCTGCACATGATGGGGTTTGGTGGACCCAGTGCCCCTCACGGCTGCCGCAAGTCTGCTGCAGATGACC T CAT GAACT GGAAGGGGT CAAGGT GACCCGGGAGGAGAGCT CAAGACAGGGCACAGGCTACT CAGAGCTG AGGGGCCCCTGGGACCCTTGGCCATCAGGCGAGGGGCTGGGCCTGTGCAGCTGGGCCCTTGGCCAGAGTC CACTCCCTTCCTGGCTGTGTCACCCCGAGCAGCTCATCCACCATGGAGGTCATTGGCCTGAGGCAAGTTC CCCGGAGAGTCGGGGTCCCCTGTGGCCCCCTCAGGCCTATGTCTGTGAGGAAGGGGCCCTGCCACTCTCC CCAAGAGGGCCTCCATGTTTCGAGGTGCCTCAACATGGAGCCTTGCCTGGCCTGGGCTAGGGGCACTGTC TGAACTCCTGACTGTCAGGATAAACTCCGTGGGGGTACAGGAGCCCAGACAAAGCCCAGGCCTGTCAAGA GACGCAGAGGGCCCCTGCCAGGGTTGGCCCCAGGGACCCTGGGACGAGGCTGCAGAAGCTCTCCCTCCCT ACTCCCTGGGAGCCACGTGCTGGCCATGTGGCCAGGGACGGCATGAGCAGGAGGCGGGGACGTGGGGGCC TTCTGGTTTGGTGTCAACAGCTCACAGGAGCGTGAACCATGAGGGCCCTCAGGAGGGGAACGTGGTAAAA CCCAAGACATTAAATCTGCCATCTCAGGC MicroRNA 4323 [MIR4323] (SEQ ID NO: 4) Homo sapiens microRNA 4323 (MIR4323), microRNA >ref|NR_036208.11:1-69 CGGGGCCCAGGCGGGCATGTGGGGTGTCTGGAGACGCCAGGCAGCCCCACAGCCTCAGACCTCGGGCAC Ring finger protein 213 [RNF213] (SEQ ID NO: 5) Homo sapiens ring finger protein 213 (RNF213), transcript variant 3, mRNA >ref|NM_001256071,3|:1-21079 GTGACCCGAGGGGCGACAGCGCGCGGCAGGCGGCGAGCTCGGGGGCCGCAGAAAATGAAACTGAAGCCGT GGTCACGTGACAGGACATGTAGTATATAGCAGGCTGCCAGCGACTCCTGCTCTTGCTTCTGGATCTGCAG GGCAGTCCCAGCAGGACCCATGGAGTGTCCTTCGTGCCAGCATGTCTCCAAGGAGGAAACCCCCAAGTTC TGCAGCCAGTGCGGAGAGAGGCTGCCTCCTGCAGCCCCCATAGCAGATTCTGAGAACAATAACTCCACAA TGGCGTCGGCCTCGGAGGGTGAAATGGAGTGTGGGCAGGAGCTGAAGGAGGAAGGGGGCCCGTGCTTGTT CCCGGGCTCAGACAGTTGGCAAGAAAACCCCGAGGAGCCCTGTTCCAAAGCCTCCTGGACCGTCCAAGAA AGCAAAAAGAAGAAAAGGAAGAAGAAAAAGAAGGGGAACAAGTCCGCTTCCTCAGAGCTGGCTTCCTTGC CCCTTTCTCCTGCCAGCCCCTGTCACCTGACTTTGCTTTCAAACCCGTGGCCTCAGGACACAGCCCTGCC CCACAGCCAAGCCCAGCAGAGTGGCCCCACTGGCCAGCCGAGCCAGCCCCCAGGCACAGCCACCACGCCA CTGGAGGGTGACGGCCTCTCCGCGCCCACCGAGGTTGGCGACAGCCCCCTGCAGGCCCAGGCTTTGGGAG AGGCAGGAGTGGCCACAGGAAGTGAGGCTCAGAGCAGCCCGCAATTCCAGGACCACACGGAAGGGGAGGA CCAGGACGCTTCCATCCCCTCTGGGGGCAGAGGCCTGTCCCAGGAGGGGACCGGTCCCCCCACCTCTGCT GGTGAAGGCCATTCTAGGACTGAAGATGCTGCCCAGGAGCTCCTGTTGCCTGAGTCAAAAGGAGGCAGCT CTGAGCCCGGGACAGAACTGCAGACCACCGAGCAACAGGCAGGGGCCTCAGCCTCTATGGCAGTTGATGC T GTAGCT GAGCCAGCCAAT GCAGTTAAAGGGGCCGGGAAGGAAAT GAAAGAGAAGACCCAGAGAAT GAAA CAGCCACCAGCAACCACTCCTCCTTTCAAAACACACTGCCAGGAAGCTGAGACCAAGACCAAGGACGAGA TGGCTGCTGCTGAAGAAAAAGTCGGTAAGAATGAACAAGGGGAGCCTGAAGACCTCAAGAAGCCAGAGGG GAAGAACAGAAGTGCAGCT GCT GT GAAAAACGAGAAGGAGCAAAAAAACCAGGAAGCAGAT GT CCAGGAA GTGAAGGCAAGCACGCTGAGCCCGGGTGGAGGAGTCACCGTGTTCTTCCACGCCATCATCTCTCTTCATT TCCCATTCAATCCTGACCTCCATAAAGTCTTCATCAGAGGAGGAGAAGAATTTGGGGAGTCAAAATGGGA CAGCAATATCTGTGAGCTGCACTACACCAGAGACTTGGGTCATGACCGCGTTCTTGTTGAAGGCATTGTC TGCATTTCCAAGAAGCACCTAGATAAATACATTCCTTACAAGTACGTCATTTATAATGGGGAATCTTTTG AGTATGAGTTCATTTACAAGCACCAGCAGAAGAAGGGCGAGTACGTCAACCGCTGTCTGTTCATAAAATC TTCACTTCTGGGCTCAGGAGACTGGCATCAGTACTATGACATAGTTTATATGAAGCCTCATGGGAGACTC CAGAAAGTCATGAACCACATCACAGACGGGCCGAGGAAGGACCTGGTGAAGGGGAAGCAGATTGCCGCTG CGCTCATGCTGGACAGCACCTTCAGCATCCTGCAGACCTGGGACACCATCAACCTGAACAGCTTCTTCAC CCAGTTCGAGCAGTTTTGCTTTGTCCTGCAACAGCCTATGATTTATGAAGGACAGGCACAGCTGTGGACC GATTTGCAGTACAGGGAGAAAGAGGTGAAGAGATACCTGTGGCAACATCTGAAAAAACACGTGGTACCAT TGCCGGACGGAAAAAGCACGGACTTTTTGCCTGTGGACTGCCCAGTGAGGAGTAAACTGAAAACAGGCCT GATTGTCCTTTTTGTAGTGGAAAAAATTGAGCTTTTATTAGAAGGCAGCCTGGACTGGTTGTGTCACCTC CTAACCTCAGATGCCAGCTCACCAGATGAGTTTCACCGTGACCTAAGCCACATCCTTGGGATACCTCAGA GCTGGCGGCTGTACCTGGTGAACCTGTGCCAAAGATGCATGGACACAAGGACGTACACCTGGCTGGGCGC CCTGCCTGTCCTGCACTGCTGTATGGAGCTGGCCCCGCGGCACAAGGATGCCTGGAGACAGCCTGAGGAC ACCTGGGCCGCTCTGGAGGGACTCTCCTTCTCACCGTTCCGGGAACAAATGCTAGATACGAGTTCCCTAC TTCAGTTTATGAGAGAGAAGCAGCATTTGCTGAGCATAGACGAGCCTCTCTTCCGGTCCTGGTTTAGTCT GCTACCTCTGAGTCACCTGGTTATGTATATGGAAAACTTCATTGAGCACCTGGGTCGTTTTCCTGCTCAT ATCCTGGACTGTCTTTCAGGGATTTACTACCGGCTTCCGGGACTTGAGCAAGTCTTGAATACGCAGGATG TTCAGGATGTTCAGAACGTTCAGAACATTTTAGAAATGCTGTTGCGACTCCTGGACACTTACCGGGACAA GATTCCCGAGGAGGCCTTGTCACCATCCTACCTGACTGTGTGTCTGAAACTGCATGAAGCCATCTGCAGC AGCACAAAGCTACTTAAGTTTTACGAGCTGCCAGCCTTATCTGCCGAGATTGTCTGCAGAATGATTAGAC TTCTATCTCTGGTGGATTCTGCAGGACAGAGAGATGAAACTGGAAATAATTCAGTCCAAACAGTCTTCCA AGGGACCCTTGCTGCTACGAAAAGGTGGCTCCGAGAAGTTTTTACAAAGAACATGCTCACATCTTCAGGT GCCTCATTCACATACGTCAAGGAAATTGAGGTCTGGAGGCGGCTGGTGGAAATCCAATTCCCCGCGGAGC ATGGCTGGAAGGAGTCGTTGCTGGGAGACATGGAATGGAGGCTCACAAAGGAGGAACCCCTCTCCCAGAT CACTGCCTACTGCAATAGTTGCTGGGACACCAAAGGCTTAGAGGACAGTGTGGCCAAGACCTTCGAGAAA TGCATCATTGAAGCCGTGAGCTCAGCCTGCCAGTCTCAGACCAGTATCCTTCAGGGGTTCTCTTACTCTG ATTTGCGGAAATTTGGCATCGTCTTGTCTGCTGTGATCACTAAGTCCTGGCCTAGGACCGCGGACAACTT CGATGACATTTTAAAGCATCTGCTCACGTTGGCAGATGTCAAGCACGTCTTCAGATTGTGTGGAACCGAC GAGAAAATACTAGCAAATGTCACAGAGGATGCCAAGAGGCTGATAGCTGTTGCCGACTCTGTGTTGACGA AAGTTGTTGGTGACCTCCTAAGTGGCACGATTTTAGTTGGACAACTGGAGCTGATTATAAAGCACAAGAA TCAGTTTCTTGACATCTGGCAACTGAGGGAAAAAAGTCTTTCACCCCAGGATGAACAATGTGCTGTGGAG GAAGCACT GGATTGGAGAAGGGAGGAACT GTTACTT CTAAAGAAAGAGAAAAGAT GT GTT GATAGT CTCC T GAAGAT GT GT GGGAACGT GAAACAT CT GATACAAGT GGACTTT GGAGTGCTTGCAGTAAGACACT CACA AGACCTCAGCAGTAAAAGATTAAATGACACCGTGACAGTGAGACTGTCCACCTCCTCGAACTCGCAGAGG GCAACGCATTACCACCTGAGCTCCCAGGTCCAAGAAATGGCTGGGAAGATAGACTTGCTCAGAGACAGCC ACATCTTCCAGCTCTTCTGGCGGGAAGCCGCAGAGCCGCTGAGTGAGCCTAAGGAGGACCAGGAAGCCGC AGAGTTGCTGAGTGAGCCCGAAGAAGAATCAGAAAGGCACATCCTTGAGCTTGAAGAGGTGTATGACTAT TTGTATCAGCCTTCTTACAGAAAGTTCATTAAGTTGCACCAGGATCTAAAGTCAGGAGAGGTCACCCTTG CAGAGATTGATGTCATCTTCAAGGACTTTGTGAATAAATACACGGACCTGGATTCAGAACTTAAGATCAT GTGCACCGTGGACCACCAGGACCAAAGAGATTGGATCAAGGACCGAGTCGAACAGATCAAGGAATACCAT CACCTGCACCAGGCTGTCCACGCAGCCAAGGTCATCTTGCAGGTCAAAGAGAGCCTGGGACTGAACGGTG ACTTCAGTGTTCTCAACACTTTACTAAATTTTACTGATAACTTCGACGACTTTCGCCGTGAAACACTGGA CCAGATCAACCAGGAGCTCATCCAGGCCAAAAAGCTGCTCCAGGACATCAGCGAGGCCCGGTGCAAGGGG CTGCAGGCTCTGTCCCTGAGAAAGGAGTTCATCTGCTGGGTCCGGGAGGCTCTTGGAGGCATCAATGAGC TGAAGGTGTTTGTGGACCTGGCCTCCATCTCAGCGGGGGAGAATGACATTGATGTGGACCGGGTGGCCTG CTTCCATGACGCTGTGCAGGGCTACGCATCCCTGCTATTTAAGCTGGACCCCAGCGTGGACTTCAGTGCA TTCATGAAGCATCTGAAAAAGCTGTGGAAGGCTCTGGATAAGGACCAGTACCTGCCCAGGAAACTGTGTG ACTCCGCCAGGAACTTGGAATGGCTGAAGACTGTGAATGAGAGTCATGGGTCTGTGGAACGCTCATCCCT GACCCTGGCCACGGCCATCAACCAAAGAGGCATCTATGTGATCCAGGCGCCCAAAGGTGGCCAAAAGATT TCCCCAGACACGGTTCTGCACTTGATCCTTCCTGAGAGCCCTGGCAGCCACGAGGAGTCACGAGAGTACT CTTTAGAGGAGGTGAAGGAGCTTTTGAACAAGTTGATGCTGATGTCTGGCAAGAAGGATCGTAACAACAC GGAAGTGGAGAGGTTTTCAGAGGTCTTCTGCAGTGTGCAGAGGCTCAGCCAGGCCTTCATCGACCTGCAC TCTGCTGGGAATATGCTGTTCAGGACGTGGATCGCCATGGCCTACTGCTCCCCCAAGCAGGGTGTGTCCC TCCAAATGGACTTTGGCTTGGACCTGGTGACGGAGCTTAAAGAAGGTGGAGATGTCACTGAGCTGCTGGC AGCCCTCTGCAGGCAGATGGAGCACTTCCTTGACAGCTGGAAGAGATTTGTGACCCAGAAGCGAATGGAG CACTTTTACCTGAACTTCTACACGGCAGAGCAGCTGGTTTACCTGAGCACTGAGCTCAGGAAGCAGCCCC CGAGTGATGCCGCCCTAACGATGCTATCCTTCATCAAAAGCAACTGCACCCTGAGGGATGTCTTAAGGGC CTCTGTGGGGTGTGGGAGTGAGGCCGCCAGGTACCGCATGAGGAGAGTCATGGAAGAGCTCCCGCTGATG CTCTTATCAGAGTTCAGCCTGGTGGACAAGCTGAGGATCATCATGGAGCAGTCCATGAGGTGCCTTCCTG CCTTCCTGCCCGACTGCCTCGACCTAGAGACCCTTGGCCACTGTCTGGCTCACCTGGCAGGGATGGGTGG GTCTCCCGTGGAGCGTTGTCTCCCGAGAGGTCTGCAGGTCGGCCAGCCCAACCTCGTCGTCTGTGGCCAC TCCGAGGTGTTGCCAGCCGCCCTGGCTGTCTACATGCAAACCCCAAGCCAGCCCCTGCCCACTTACGATG AGGTGCTGCTCTGCACCCCGGCAACCACCTTTGAGGAGGTGGCACTGTTGCTGCGCCGCTGCCTGACCCT GGGCTCCCTGGGGCACAAGGTCTACAGCCTGCTGTTCGCAGATCAGCTGAGCTACGAGGTGGCACGCCAA GCGGAGGAGCTTTTCCACAATCTGTGCACGCAGCAGCACCGAGAAGACTACCAGCTCGTCATGGTCTGTG ATGGGGACTGGGAGCACTGCTACCTCCCCTCTGCCTTCAGCCAGCACAAGGTCTTCGTCACCCCCCAGGC ACCCCTCGAGGCCATCCAAGCCTACCTGGCAGGTCACTACCGGGTCCCGAAGCAGACCCTGTCGGCGGCA GCCGTGTTCAATGACCGGCTGTGTGTTGGGATCGTGGCCTCGGAGCGAGCAGGTGTTGGAAAGTCTCTGT ACGTGAAGAGGTTGCACGACAAAATGAAGATGCAGTTAAACGTGAAAAATGTGCCTCTGAAAACAATTCG ACTGATCGACCCTCAGGTGGATGAGAGCCGAGTCCTGGGCGCCCTGCTGCCCTTCCTGGATGCGCAGTAT CAGAAGGTCCCCGTGCTCTTTCACCTGGACGTGACCTCCTCAGTGCAGACTGGAATTTGGGTGTTTCTTT TCAAGCTCCTCATTTTACAATACTTAATGGATATAAATGGGAAAATGTGGCTTCGGAACCCCTGCCATTT GTATATCGTTGAAATCCTGGAAAGGAGGACGTCAGTGCCGTCGAGGAGCTCTTCAGCGCTGCGTACACGT GTACCCCAGTTCAGTTTTCTTGACATCTTCCCAAAAGTCACCTGCAGGCCTCCCAAAGAGGTGATAGACA TGGAGCTGAGTGCCCTGAGGAGTGACACAGAGCCTGGGATGGATCTGTGGGAGTTCTGCAGCGAAACTTT C CAAAGAC C T TAC CAGTAT T TAAGAC GAT T CAAT CAAAAC CAAGAC C TAGACAC GT T T CAGTAT CAAGAA GGCTCTGTCGAAGGCACCCCGGAGGAATGCCTCCAGCATTTCCTGTTTCACTGCGGGGTAATAAACCCAT CCTGGTCAGAGCTTCGGAACTTTGCTCGGTTCCTGAATTATCAGCTCAGAGATTGTGAGGCCTCTCTCTT CTGCAATCCGAGTTTTATTGGCGACACACTGAGGGGCTTCAAGAAGTTCGTGGTGACCTTCATGATCTTT ATGGCAAGAGATTTTGCCACACCATCACTCCACACCTCTGACCAAAGCCCGGGGAAGCACATGGTCACCA TGGATGGGGTTAGGGAAGAAGATCTAGCGCCCTTCTCCCTCCGGAAGAGGTGGGAGTCGGAGCCTCACCC ATACGTTTTCTTCAATGACGACCACACAACCATGACATTCATCGGCTTCCATCTGCAGCCCAACATCAAC GGCAGTGTCGATGCCATCAGTCACTTGACTGGGAAGGTCATCAAGAGAGACGTCATGACCAGGGACCTGT ACCAGGGCCTGCTGCTCCAGAGGGTGCCCTTCAATGTCGACTTTGATAAACTGCCCAGACACAAGAAACT TGAGAGGCTCTGCCTGACCTTAGGGATCCCCCAGGCCACCGACCCCGACAAAACGTATGAGCTCACAACC GACAATATGCTTAAAATCCTTGCCATCGAGATGCGGTTCCGGTGTGGGATCCCGGTTATCATCATGGGAG AAACTGGCTGTGGGAAAACCAGGCTTATTAAATTCCTTAGCGACCTGCGGCGTGGTGGTACCAATGCTGA CACCATAAAGCTGGTCAAGGTGCACGGAGGAACAACTGCAGACATGATCTACTCCAGAGTCAGGGAGGCT GAAAATGTGGCCTTCGCCAATAAGGACCAACATCAGTTGGACACCATCTTGTTTTTTGATGAAGCCAACA CAACGGAAGCTATAAGCTGTATCAAAGAAGTCCTGTGTGATCATATGGTGGATGGCCAGCCTCTGGCTGA GGACTCTGGCCTGCATATTATAGCTGCCTGCAATCCATACCGGAAGCACTCTGAGGAGATGATCTGCCGT TTGGAGTCAGCTGGTTTGGGCTACAGGGTTAGTATGGAGGAGACGGCCGACAGGCTGGGCTCCATTCCTC TGAGGCAGCTGGTATACCGGGTCCATGCTCTGCCCCCGAGCCTGATTCCTCTGGTGTGGGACTTTGGACA ACTGAGTGACGTTGCTGAAAAGCTCTACATCCAGCAGATTGTCCAGAGACTGGTTGAGTCCATCAGCCTA GATGAAAACGGGACTCGCGTGATCACAGAAGTCCTCTGCGCCTCTCAGGGTTTCATGAGGAAAACAGAAG ATGAGTGCAGCTTTGTCAGCCTCAGGGACGTGGAGCGCTGTGTGAAAGTTTTCAGGTGGTTCCACGAGCA CAGCGCGATGCTCTTAGCGCAGCTGAATGCCTTTCTCTCCAAGTCCAGCGTCAGCAAAAATCACACCGAG AGAGATCCCGTCCTCTGGTCGTTGATGCTGGCCATCGGGGTGTGTTACCATGCCTCTTTAGAAAAGAAAG ACTCATATCGGAAAGCCATCGCCAGGTTCTTTCCGAAACCGTATGACGACAGCAGGCTGCTTCTGGATGA AATAACACGGGCACAGGATCTTTTTCTGGACGGCGTACCTCTGAGGAAAACCATCGCCAAGAACTTGGCC TTGAAGGAGAACGTCTTCATGATGGTCGTCTGCATCGAGCTGAAGATTCCCCTCTTCCTGGTGGGGAAGC CCGGCAGCTCCAAGTCTCTCGCCAAGACCATCGTGGCAGACGCCATGCAGGGCCCGGCTGCCTACTCAGA TCTCTTCCGCAGCCTGAAGCAGGTCCACCTGGTGTCCTTCCAGTGCAGCCCGCACTCCACCCCACAGGGC ATCATCAGCACCTTCCGGCAGTGCGCCCGCTTTCAGCAGGGGAAGGACCTGCAGCAGTACGTCTCTGTGG TGGTGTTAGATGAGGTGGGGCTGGCGGAAGACTCACCCAAAATGCCCCTGAAGACTCTGCACCCGCTGCT GGAAGACGGATGCATTGAAGACGATCCCGCCCCCCACAAAAAGGTCGGCTTCGTGGGCATCTCCAACTGG GCCCTTGACCCTGCCAAGATGAACCGGGGCATTTTTGTGTCACGTGGCAGCCCCAACGAGACAGAGCTCA TAGAGAGCGCCAAGGGCATCTGCTCCTCAGACATCCTCGTCCAGGACCGAGTCCAAGGGTACTTTGCGTC CTTTGCCAAAGCCTACGAAACGGTGTGTAAGCGCCAGGACAAGGAATTCTTCGGGCTTCGTGACTACTAC AGCCTCATCAAAATGGTCTTTGCTGCAGCAAAGGCTTCAAATAGAAAGCCTTCCCCGCAAGACATTGCAC AGGCTGTCCTTAGGAACTTCAGTGGCAAGGATGACATCCAAGCTTTGGACATCTTTCTGGCCAATTTGCC CGAGGCCAAGTGCTCAGAGGAAGTCAGCCCCATGCAGCTGATCAAACAGAACATCTTTGGGCCTTCTCAG AAGGTGCCGGGTGGAGAGCAGGAAGATGCTGAGTCCCGCTACTTACTCGTGCTGACCAAAAACTACGTGG CACTGCAGATCCTGCAGCAGACATTCTTCGAGGGGGACCAGCAGCCGGAGATTATTTTTGGTTCTGGTTT CCCCAAGGACCAAGAGTACACCCAGCTCTGCAGAAACATCAATCGTGTGAAGATCTGCATGGAAACAGGC AAGATGGTGTTGCTTCTCAACCTGCAGAACCTCTACGAGAGCCTCTACGACGCACTCAACCAGTACTACG TCCACCTCGGCGGCCAGAAGTACGTGGACCTCGGTCTGGGGACCCACCGCGTCAAATGTCGGGTTCACCC CAACTTCCGCCTGATTGTCATTGAAGAGAAAGACGTCGTGTACAAACACTTTCCCATCCCCCTCATTAAC CGGCTGGAGAAGCACTATCTGGATATCAACACGGTGCTGGAGAAATGGCAGAAGAGCATCGTGGAGGAGC TCTGTGCGTGGGTGGAGAAGTTCATCAATGTCAAAGCACATCATTTCCAGAAGAGGCACAAATACAGCCC CTCTGACGTCTTCATCGGCTACCACTCGGACGCCTGCGCGTCTGTGGTGCTGCAGGTCATAGAGAGGCAG GGTCCCCGGGCCTTGACGGAGGAACTTCACCAGAAGGTGTCTGAGGAGGCCAAATCGATCCTGCTGAACT GCGCTACGCCCGATGCCGTGGTCCGGCTGAGCGCCTACTCGCTGGGCGGGTTCGCAGCGGAGTGGCTGTC GCAGGAGTACTTTCACAGACAGAGGCACAACTCCTTTGCAGATTTCCTTCAGGCACACCTGCACACGGCA GACCTGGAGCGCCACGCCATCTTCACAGAGATCACCACTTTCTCCAGGCTGCTAACAAGTCACGACTGTG AAATTTTAGAATCAGAGGTCACAGGCAGGGCTCCGAAACCCACACTCCTGTGGCTGCAGCAGTTTGACAC CGAGTACTCATTCCTCAAAGAAGTCCGAAACTGTTTAACGAATACAGCCAAATGTAAAATCCTCATTTTT CAGACAGATTTTGAAGATGGAATCCGTAGCGCCCAGCTCATTGCCTCAGCTAAGTATTCTGTTATAAATG AAATCAACAAAATACGAGAAAATGAGGACCGTATCTTCGTCTATTTCATCACAAAACTGTCCCGGGTGGG AAGAGGAACAGCCTATGTGGGCTTCCACGGAGGGCTGTGGCAGTCTGTCCACATCGATGACCTCCGGAGA TCCACCCTCATGGTTTCTGATGTGACCAGGCTGCAGCATGTCACCATCAGCCAGCTGTTCGCGCCCGGAG ACTTGCCTGAGCTGGGCTTGGAACACCGGGCGGAAGACGGCCATGAGGAGGCGATGGAGACGGAGGCCAG CACAT CAGGGGAGGTGGCAGAGGT GGCAGAGGAGGCCAT GGAAACAGAAAGTTCT GAGAAGGT GGGAAAG GAAACCTCTGAACTCGGAGGCAGTGATGTGTCGATCCTGGACACCACCAGGCTGCTGAGAAGCTGTGTGC AGAGCGCCGTGGGCATGCTCAGAGACCAGAACGAGAGCTGCACGCGCAATATGCGGAGGGTGGTGCTCCT CCTGGGCCTCTTGAATGAGGATGACGCGTGCCACGCCTCTTTCTTGCGGGTATCCAAGATGCGCCTCAGT GTCTTTTTAAAGAAGCAAGAAGAGAGCCAGTTTCACCCTCTGGAGTGGTTGGCAAGGGAAGCCTGCAACC AGGACGCTCTCCAGGAGGCGGGCACATTCAGGCACACCCTCTGGAAGCGGGTCCAAGGTGCTGTCACCCC TCTGCTGGCGAGCATGATATCATTCATCGACAGAGACGGCAACCTAGAGTTACTGACCAGGCCAGATACT CCGCCCTGGGCAAGAGATCTTTGGATGTTTATTTTCAGTGACACGATGCTTCTGAACATTCCTCTTGTGA TGAATAATGAAAGACATAAAGGTGAGATGGCCTACATCGTGGTGCAGAACCACATGAACCTTTCCGAGAA CGCTTCCAACAACGTCCCTTTCAGCTGGAAAATCAAGGACTATCTGGAGGAGCTGTGGGTCCAGGCTCAG TACATCACAGACGCAGAAGGACTGCCCAAGAAGTTCGTGGACATCTTTCAGCAGACTCCTCTGGGCAGGT TTCTTGCCCAGCTCCATGGAGAGCCGCAGCAGGAACTTCTTCAGTGTTACTTGAAGGATTTCATTCTCTT GACCATGCGTGTGTCAACGGAGGAGGAATTAAAGTTTCTGCAGATGGCTCTGTGGTCCTGCACTAGGAAA CTGAAAGCGGCGTCAGAAGCGCCCGAGGAAGAGGTTTCCTTACCGTGGGTGCACCTTGCCTACCAGCGTT TCAGAAGCCGTCTGCAGAACTTTTCCAGAATCCTGACCATCTACCCTCAGGTTCTCCACAGCCTGATGGA AGCCCGTTGGAACCATGAGCTGGCTGGATGTGAGATGACCCTGGACGCATTTGCCGCAATGGCCTGCACG GAGATGCTGACAAGAAACACCCTGAAGCCCAGTCCCCAGGCGTGGCTACAGTTGGTGAAGAATCTTTCCA TGCCGCTGGAGCTCATCTGCTCCGATGAGCACATGCAAGGCAGCGGGAGCCTGGCCCAGGCTGTCATCAG GGAAGTCAGAGCCCAGTGGAGTCGGATTTTCTCCACCGCACTCTTCGTGGAGCACGTGCTCCTAGGAACC GAGAGCCGCGTCCCCGAGTTACAGGGGCTGGTGACCGAGCACGTCTTCTTACTAGACAAGTGTCTTCGAG AGAACTCTGACGTGAAGACGCACGGGCCTTTTGAGGCCGTGATGCGCACTCTCTGTGAATGCAAGGAGAC AGCCAGCAAGACCCTCAGCAGGTTTGGGATTCAGCCGTGCTCCATCTGCCTGGGAGATGCAAAGGACCCC GTCTGTCTGCCCTGCGACCACGTGCACTGCCTGCGCTGCCTCAGGGCCTGGTTTGCCTCAGAGCAGATGA TATGCCCCTACTGTTTAACTGCCTTGCCAGACGAATTCTCTCCAGCTGTTTCCCAAGCGCACAGGGAAGC CATTGAAAAGCATGCCCGCTTCCGGCAGATGTGCAACAGTTTCTTCGTAGACCTGGTGTCCACCATTTGC TTCAAGGACAACGCTCCGCCTGAGAAGGAAGTGATTGAGAGCCTGCTCTCTCTCCTCTTCGTCCAAAAGG GGCGCTTAAGAGATGCTGCCCAGAGACACTGTGAACACACAAAATCTCTCTCTCCATTCAATGATGTTGT GGATAAGACTCCTGTCATCCGCTCAGTGATACTGAAACTGCTTTTGAAGTACAGCTTTCATGATGTAAAA GAT TAT AT T C AG GAAT AT TTGACCCTGT T AAAAAAGAAAG CAT T C AT AACT GAAGAT AAAAC T GAACT GT ACATGCTCTTCATCAACTGCCTGGAGGATTCAATACTTGAGAAGACCAGTGCTTACTCCAGAAATGATGA ACTGAACCACCTAGAAGAGGAAGGTCGTTTCCTTAAGGCATATTCTCCAGCAAGCCGGGGCCGAGAGCCT GCCAACGAGGCCTCGGTTGAATACCTGCAAGAGGTGGCCCGGATCCGCCTCTGCCTCGACAGAGCTGCAG ATTTCCTCTCGGAGCCTGAGGGAGGCCCAGAGATGGCCAAGGAGAAGCAGTGCTACCTGCAGCAAGTCAA GCAGTTCTGTATCCGGGTGGAGAACGACTGGCACCGGGTGTACCTGGTGCGGAAGCTCAGCAGCCAGCGG GGGATGGAGTTCGTGCAGGGCCTCTCCAAGCCCGGCCGCCCGCACCAGTGGGTGTTTCCCAAGGACGTTG TCAAGCAGCAGGGGCTGCGGCAGGACCACCCAGGCCAGATGGATAGGTACCTGGTGTACGGCGATGAATA CAAGGCTCTCCGTGATGCTGTGGCCAAAGCTGTCCTCGAGTGCAAGCCACTGGGCATTAAGACTGCTCTG AAGGCCTGCAAGACCCCCCAAAGCCAGCAGTCAGCCTACTTCCTGTTAACACTGTTTAGAGAGGTGGCTA TTTTGTACAGATCCCACAATGCAAGCCTCCACCCCACGCCAGAGCAATGTGAAGCTGTGAGCAAATTCAT TGGCGAATGCAAGATCCTTTCACCTCCTGATATCAGCCGTTTTGCAACATCGCTCGTGGACAATTCTGTG CCATTGTTGAGGGCGGGGCCTAGTGACAGCAACCTTGATGGAACGGTGACAGAAATGGCCATTCATGCTG CAGCCGTCCTTCTGTGTGGACAGAATGAACTCTTGGAGCCCCTAAAGAATCTGGCCTTCTCCCCAGCCAC CATGGCGCATGCTTTTCTTCCAACCATGCCTGAAGACTTGCTGGCTCAAGCTCGGAGGTGGAAGGGTCTG GAGCGAGTCCACTGGTACACTTGTCCCAACGGCCATCCTTGCTCCGTGGGAGAGTGTGGCAGGCCGATGG AACAGAGCATCTGCATTGACTGCCATGCGCCGATTGGAGGCATTGACCACAAACCTCGGGACGGCTTTCA TCTGGTCAAAGACAAGGCAGACAGAACGCAGACCGGCCACGTGCTGGGCAACCCGCAGCGGAGAGACGTG GTGACATGTGACCGAGGGCTGCCCCCAGTGGTCTTCCTCCTTATCCGGCTACTCACTCACTTGGCTCTGC TTCTGGGAGCGTCCCAGAGTTCCCAGGCTCTGATAAACATCATTAAGCCTCCAGTGAGGGATCCAAAAGG CTTTCTGCAGCAGCACATCCTGAAGGACCTGGAGCAGTTGGCCAAGATGCTGGGACACAGTGCCGACGAG ACCATCGGCGTGGTCCACCTCGTCCTGCGCAGGCTTCTCCAAGAGCAGCACCAGCTCTCTAGCAGAAGGC TTTTAAATTTTGACACAGAATTGTCAACTAAAGAAATGAGGAACAACTGGGAAAAGGAAATCGCAGCTGT GATTTCTCCTGAACTGGAGCATCTAGATAAAACCCTTCCCACCATGAATAATCTCATCAGCCAAGATAAG CGTATCAGCTCTAACCCTGTGGCCAAAATAATATATGGTGACCCAGTGACCTTCCTGCCCCACCTGCCCC GGAAAAGTGTGGTCCATTGCTCTAAGATTTGGAGCTGCAGGAAAAGAATTACAGTTGAGTACCTCCAGCA CATTGTGGAACAGAAAAATGGCAAAGAAAGAGTGCCCATCCTCTGGCATTTCCTGCAGAAGGAAGCAGAG CTGAGGCTGGTAAAGTTCCTGCCTGAGATTTTGGCCTTGCAAAGGGATCTAGTGAAGCAGTTCCAGAACG TCCAGCAAGTTGAATACAGCTCCATCAGAGGCTTCCTCAGCAAGCACAGCTCAGATGGGTTGAGGCAGCT GCTTCACAACAGGATCACAGTCTTTCTGTCCACATGGAACAAACTGAGGAGATCGCTTGAGACGAACGGT GAGATCAACCTACCCAAAGACTACTGCAGCACTGACTTGGATCTGGACACTGAGTTTGAGATCCTCTTGC CACGCCGACGGGGCCTGGGCCTCTGTGCTACCGCTCTCGTCAGCTACTTGATTCGCCTACACAATGAAAT TGTCTACGCCGTGGAAAAACTCTCCAAGGAAAACAACAGCTATTCCGTGGATGCCGCCGAGGTCACTGAA CTGCATGTCATCAGTTATGAAGTGGAGCGGGACCTGACTCCACTGATTCTCTCCAACTGCCAGTACCAGG TGGAGGAGGGCAGAGAGACCGTGCAGGAGTTCGATCTGGAGAAGATTCAGCGGCAGATCGTCAGCCGCTT CCTCCAGGGCAAGCCCCGGCTGAGCCTCAAGGGAATACCCACTCTGGTGTACAGACACGACTGGAACTAT GAACATCTCTTTATGGACATCAAGAACAAAATGGCACAGGACTCCCTCCCCAGCTCGGTCATTAGTGCCA TCAGTGGACAGCTGCAGTCCTACAGCGATGCCTGTGAAGTGCTGTCTGTCGTAGAAGTCACTCTGGGGTT TCTGAGCACAGCTGGTGGGGATCCAAACATGCAGCTGAATGTGTATACTCAAGACATCCTGCAAATGGGT GATCAGACGATTCACGTGTTAAAGGCCTTAAACAGATGCCAGTTAAAACACACCATTGCCCTCTGGCAGT TCCTGTCTGCTCATAAGTCTGAACAGCTGCTGCGGCTGCACAAAGAGCCATTTGGGGAAATCAGTTCAAG GTACAAAGCGGATCTGAGCCCGGAAAATGCTAAGCTCCTCAGCACATTCCTAAATCAGACTGGCCTAGAC GCCTTCCTGCTAGAGCTGCACGAAATGATAATCTTGAAACTAAAGAACCCCCAAACCCAAACCGAGGAGC GCTTCCGCCCTCAGTGGAGCCTGAGAGACACTCTCGTAAGTTACATGCAAACTAAAGAAAGTGAAATTCT TCCTGAAATGGCATCTCAGTTCCCAGAAGAGATACTGCTCGCCAGCTGTGTCTCAGTGTGGAAAACAGCT GCTGTGCTGAAATGGAATCGAGAAATGAGATAGAATTATTTCCTCAGCTATCTTTGGATGACTTTGGAGA GAAGACTCCTCTCTCCTCGTCTGCGGCGTGGACTTGATCATGGACTGGTGCCTTTGCATTCAGAAGGAGA GCTGTCAGCGTAGCACCGAATTCAAGACCAAGGCGTGCTACCTGAGCTGACAGCTTTTTGAAAGCCGAGC TGTTTCTGAACCATGTACATACATGTTCTGAAACTTTCTCATCATTTTATGAGTACTGTTCATTGAGAGA TGACAATGAAGATTAGATGAAATTGGAAATAAACCAACATTGTTTACATTCCAGGAGACTTGTAGCTCAG CCACACACGCAGTAATGACCTGTGCCCGTTCGCCTCTGGCACTGCCCACCCCTCTTTTTTTTTTTCTTCT AATTCTGTACTCACAAAAGAGAATCTCATTTTCTTCTTTCTTCCATTCCCTTAAATTCTGAGTACTGTAC ATATATTTCTGGGTTCCCACGATGATGTGAAAAACTACCAGACTGTTTTTTGTCTTCTCACAAAGACAAG AAAAATCAGGGCATTTTGTGAGTGCCTTAAGATCAAACTAACAAGATCTGACCCTCTCCCCTCACAGTGA GCCACTGCCCCACTTCAGAGGGTAAGAGCCAAAAGCCTCATTGTGAAAGGCACTGGACTTGGACCAGGGA CACCATCAGGGCCTTGGTTTTCTCACGCATAAAATGGAGAGTGGATTAATCGCCAAAGATTCTTCTGATC TGACATTTTGAAATTGTGAGAGAAACTAGATGACTGTAAACTTGGTCACAGGCCTGGTTCTGGCAGTTCT TTGCGGACTTTTTTCTAGCATTATGCCAAATAAACATGCAGTCTCAGTGTGCTCTCGCATGTATGAATAT CTAGTCCTTTCTGTGGTTCTCAGCCAAGACATAAAAACTAGGACTCAGAGCACATACAAAACCAGTTATG TTTCGGAAAGAGGGAAAAGAGTCCCCGAGCCCGGATCTTGTGCTGCTTTTCTCACTGACGTGTTGCCTTT TTTCTTTACAAAATCTGCTTTGATACTTAGGACCTCTCTGGACTAATTTCTCTTCCTAGACAGCTCAGCA CAGCTATTGATATGTTAGAGGCAGTATCCTTAATATTCATTCTAAATGAGTTAACGACTTAACTTGAAAT TGGGCCTAAGGAGTGAGAACTACAAAAATACAAAATGCTTGTCCAGGACTCAGCCATGCACACCTTGAGC AGCGCCGGCAGGAGGCACGGAAGGAACTGTGCTCCGTTCTCCTCACTGTCATGGTGCCACCAGTGTCTGA TGAAGGGCAGAGTGACCCAGACTGCAGGCAGTAACTGACTTCACACAGTCCCTGGCATTTAGTCATCTGT GATTGTTTTATCACTCTGGACTGTGCAGAGCCACCTGCCACCGAGATCTGCATTCCGACTGCCTATGAAC GGGTGTGGGGGCCGGGGGCTGGCTTGCTGAAGTCTTCAACTTGCACTCGGAGCTCCTTTGATACCTCAGA GCTGGCTGTCAGGTGGCAGCTCACACCCAGACTCACTGGCCACACCTCAGCAGGGGGGGAGTCGAGTGTC AGTCTCTTTCTGTGAAGGCTTTTTTTTTCCTTTGGCCTGGGAATTTTTCCCATTTTTATGAAGGGGTTTT AAATTGTTTCATTTTGTGTGCTGTGCTTCAAAGCCTTAACTGTCAAATCTTGCATTATCTTGTTTGTACA GAAATATACTGGCCTAGCAGAGGCAAAAAAAAAAAAAATGAATTTTATTTTACTTGTCACACCTGTCTTA ATAAACTGGAGTTTTGCTGCTAAAGAACTCTTCTCTCTGGGGGCAGAGCTTCTATTTATGGCACATAGAC ATCAGCTAGGCTTTTGGGAATCGTTTGTGTTCTTTGTGGAAATGTCCTTTAGAAGCACCCATGAAGTAGT GTGTTCAGACTGTGCACACAGAAAACAGGCTCTGCCTTCACATGTGAGACGGTGGACTTTTCCTCTGGAC AAAATGACAGCATCCTGGCGACTCCACAGTGGAGCTGAGCGCCACTCCCTGTAGCCGATCTGGGACTGAA ACGCTTACACCTCTGCCTCAGAAGGAGTCCCCCATGCCCTGCCTGAAATGACTTCACTGGACACAGCGGG GCTGCAGCTAACGGGGTACAGGTAGGAGCTAACTAACTTCACCCCTGAGTCCACTTGCGGGGTAAGAGAT AAACAGTAACCCTTCCAGGAGCCCACTGACGTTGGAGTGCTAAAAATGCCCCTTCAGGGGGAAAACTGCA TTTTCTCTTCCAAAAAGGAAAGGTTCTTCCAGGCGAGAAACCTGTGGTCTAGAACCACAGCAAGAAGAGG AGGCATGCTGGCCTGCACCGGAAGACTCACTTTGTCTGCCCTGCGCCAGCCTCACCTCACCCTGCAGTTC CCGTTTCCGCCATGGATGCCTCATCACCAACCCTGACCTTCCCCCTCCCAACCCTTTATTCATCCTCACT CCCACTCATACCCGCCTCCCTGGACAGTTCCCTGCTGCAGAGTTCTTTCTGCTTTCAGCCCTACCTTGGT GGTGATTTACCTGAAAATCTTCACAACTGATCATTATCTCCTTCTCTTTGAGACCTGACTGAAAAAATTA GGTGTGCACACCTGTAATCCCAGCTACCTGGGAGGCTGAGGTATGAGGATTGCTTGAGCCTGGGAGTTTG AGGCTGCAGTGAGCTATGATTGCACCACTGCACTCCAACCTGGGTGACAGAGTGAGACCCTGTCTCAAAA AAAAATAAAATAAGGACCTTATTGTGTGTGCAAATTATTTGCCTTTCATGAATAGTTATGAGACTTTGTG CATGTGTTAACCGAGACGGCCCTGAGCTATTTCAGTTACTCCTGTATGTTCAGGCAATTCCAATTTAGAA ACAAACTTCCAGTGATAAATATTGGGAAGAAAATGTCAGCTCAGGGCCAAGTAGGTAAAGCCACCCAAGT CTAGCCATGCACACAGGCACTAGTGTGCTGAAGGAAAACACCTCTGATTTGATACATAAAGTGTGAAGGT TGCATAACTGGGGAGGTGGCTGGAGAAAACCTGAGCCTCGAGCTGAGTAACAATCCAAGCCATGATTAGC ATTTATATTGCATTCTGCATTATTCTCATCCTATCACGTTCATTAAGTTCAACCATTACCATTACGAATC CCGTTTCCAGCTGGAAAAACAAGCGCCAGGAAAGTGCATTTCCCCCCCACCCCCCCCCCCCAACCAGAGC AACTTTGGTCAGAAGTCACACCTAATCACCCTCTCTGCAGGATATGCTGGGGATAGCCCTTTTGAGGAGG TGGGGGTGTATGCGAAAGCAACCTGGGGCCAAAGCAGGGGGAAGCAGGAGAGACTGGAGGGGCCCTGGGT CCAGCAAGTGCTCCACCAGGCTGCCCCTGCTCAGGTGCTCCACAGCCCATAAAAAACGCCTTCAGCCCTG CTGTGCTTCCCCTCACACCTAAACTTACTATAAGGGTCCTACCTTCCCCAGTATTCCTTCATGATCACTG CTACCTGTGCCCTGAAGCGCAACAGAACCTCAACCCAGAGGACAGCCACTTCCTCAGAACCCACGATCTT TGCCCCATCGCCACAGGTCCATGAGTGACTCCTAATTACCAAATCCAAGGGCTCCTTCACCCTGACCACT CTGGAACCCAACAAAGCTGACACACAGCCCAGCCCAGACAGGCCTGAATGTGGGTCCTGGCTCTCCCATT TGCCAGTTCTACAGCCATGGGCAAGTGGCTCGACCTCTCTAAGCTTCAGTTTCCTCGTCTGTGAAATATC TGTCAGTGCTCTTAAGGGAGGAGACCACCCCACATATTGTCTTACGCCCAATTTCTGCCTCCAAAGAGAG AAGTAAAAACTAAAAGGCAGAAATGAAATCCACAAGCAGACAGCCTGCACCACACCCTGGGCCTGGTAGT TAAAGATCGACGCCTGACCTAATCAGTGATGCTATCTATAGATTACAGACATTATATAGAAAAGCACCGT GAAAATCCCTGTCCTGTTCTGTTCCGTTCTAATTACGGGTGCATGCAGCCCCCAGTCACGTACCCCCTGC TTGCTCAATCAATCACGACCCTCTCACGCAGACCCCTTAAGAGTTGTGAGCCCTTAAAAGGGACAGGAAT TGCTCACTCGGGGAGCTCAGTTGTTGGAGACATGAGTCTTGCCGAAGCGCCCGGCTGAATAAAGCCCTTC TTTAACTCGTGTCTGAGGGGTTTTCTCTGCAGCTTGTCCTGCTACATTTCTTGGTTCCCTGACCAGGAAT CTAGGTGATTAATGGATGGTTGAGGCAGCCCCTCAGACGGCTTAGGCCTGCCCTGTGGAGCATCCCTGTG GAGGACTCCAGCCAGCTTGAGCGACATGGATCCTGAGAGCGCTCTCAGGCAGGCAATTGCCCCAGTGGAA TGCCTGGCCAGAGCAGTGTGTAGCAGGCCCCCGAGGAGGATCAACGCAGTGGCTGAACACCGGGAAGGAA CTGCCACTTGGAGTCCAGACATCTGAAACTTGGTAAGACTGGTCTTTGGAACTTGCCCACTCCATTTGAG GGGAAGCGTGGCCTGATCACCCACGGCGTGCCTGTACTGGCACTCTGGTTTTTGTTTTTGACTTGACTTG GATTGCTTGATACTTTGGTTTTGGTTTTGACCTGACTTGGATATCCTGATACTCTGATTTTGGTTTTGAT TTTGGTTTGGTGTAAACTGTAAAAGTGTGTGTGTGCCCTTTTTACCTGTTCTTTGTTTTGTGGTATGCGT GTAGGGTGAGCGTAATGTTTTGTCTTGAAGAAGCATGGGTCAAGCACAAAGTAAGACCACCCCACTAGGA ACTATGCTGAAAAATTTCAGGAAAGGATTTAAGGGAGACTATGGAGTACTATGACACCAGGAAAACTTAA AACTTTGTGTAAGATAGACTGGCCAGCATTAGAGGTAGGTTGGCCATCAGAGTGAAGCCTGGACAGGTCC CTTGTTTCAAAGGTATGGCACAAGGTAACCTGTAAGCCAGGGTACCCACACCAGTTCCCGTACACAGACA CTTGGTTACAGCTGGTTTTAGACTCCCCACCCGCCCGCCCCAACAGTGGTTGAGAGAACAGCAGCATAAG T GGCT GGCAGAGGCAAGGAAAGACCAGCAGAGAGAGAAAGAGGAAGAGACAGACAAAGAGGGAGT CAGAG AGAGAGAGAGAAAGAGACAGAGGCAAAAGGAAAGTCAAAGAGAAAGAGACAGAAAATCAAGAGGAAAAAA AGAAAAAAA Actin gamma 1 [ACTG1] (SEQ ID NO: 6) Homo sapiens actin gamma 1 (ACTG1), transcript variant 2, mRNA >ref|NM_001614.5|:1-1919 CTCAGTCGCCGCTGCCAGCTCTCGCACTCTGTTCTTCCGCCGCTCCGCCGTCGCGTTTCTCTGCCGGTCG CAATGGAAGAAGAGATCGCCGCGCTGGTCATTGACAATGGCTCCGGCATGTGCAAAGCTGGTTTTGCTGG GGACGACGCTCCCCGAGCCGTGTTTCCTTCCATCGTCGGGCGCCCCAGACACCAGGGCGTCATGGTGGGC ATGGGCCAGAAGGACTCCTACGTGGGCGACGAGGCCCAGAGCAAGCGTGGCATCCTGACCCTGAAGTACC CCATTGAGCATGGCATCGTCACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAACGA GCTGCGCGTGGCCCCGGAGGAGCACCCAGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACAGA GAGAAGATGACTCAGATTATGTTTGAGACCTTCAACACCCCGGCCATGTACGTGGCCATCCAGGCCGTGC TGTCCCTCTACGCCTCTGGGCGCACCACTGGCATTGTCATGGACTCTGGAGACGGGGTCACCCACACGGT GCCCATCTACGAGGGCTACGCCCTCCCCCACGCCATCCTGCGTCTGGACCTGGCTGGCCGGGACCTGACC GACTACCTCATGAAGATCCTCACTGAGCGAGGCTACAGCTTCACCACCACGGCCGAGCGGGAAATCGTGC GCGACATCAAGGAGAAGCTGTGCTACGTCGCCCTGGACTTCGAGCAGGAGATGGCCACCGCCGCATCCTC CTCTTCTCTGGAGAAGAGCTACGAGCTGCCCGATGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGG TGTCCGGAGGCGCTGTTCCAGCCTTCCTTCCTGGGTATGGAATCTTGCGGCATCCACGAGACCACCTTCA ACTCCATCATGAAGTGTGACGTGGACATCCGCAAAGACCTGTACGCCAACACGGTGCTGTCGGGCGGCAC CACCATGTACCCGGGCATTGCCGACAGGATGCAGAAGGAGATCACCGCCCTGGCGCCCAGCACCATGAAG ATCAAGATCATCGCACCCCCAGAGCGCAAGTACTCGGTGTGGATCGGTGGCTCCATCCTGGCCTCACTGT CCACCTTCCAGCAGATGTGGATTAGCAAGCAGGAGTACGACGAGTCGGGCCCCTCCATCGTCCACCGCAA ATGCTTCTAAACGGACTCAGCAGATGCGTAGCATTTGCTGCATGGGTTAATTGAGAATAGAAATTTGCCC CTGGCAAATGCACACACCTCATGCTAGCCTCACGAAACTGGAATAAGCCTTCGAAAAGAAATTGTCCTTG AAGCTTGTATCTGATATCAGCACTGGATTGTAGAACTTGTTGCTGATTTTGACCTTGTATTGAAGTTAAC TGTTCCCCTTGGTATTTGTTTAATACCCTGTACATATCTTTGAGTTCAACCTTTAGTACGTGTGGCTTGG TCACTTCGTGGCTAAGGTAAGAACGTGCTTGTGGAAGACAAGTCTGTGGCTTGGTGAGTCTGTGTGGCCA GCAGCCTCTGATCTGTGCAGGGTATTAACGTGTCAGGGCTGAGTGTTCTGGGATTTCTCTAGAGGCTGGC AAGAACCAGTTGTTTTGTCTTGCGGGTCTGTCAGGGTTGGAAAGTCCAAGCCGTAGGACCCAGTTTCCTT TCTTAGCTGATGTCTTTGGCCAGAACACCGTGGGCTGTTACTTGCTTTGAGTTGGAAGCGGTTTGCATTT ACGCCTGTAAATGTATTCATTCTTAATTTATGTAAGGTTTTTTTTGTACGCAATTCTCGATTCTTTGAAG AGATGACAACAAATTTTGGTTTTCTACTGTTATGTGAGAACATTAGGCCCCAGCAACACGTCATTGTGTA AGGAAAAATAAAAGT GCT GO C GTAAC CAA The primers and probes that were designed in this study are shown in Table 2. Primers and probes targeting the Ring finger protein 213 (RNF213) and Actin gamma 1 (ACTG1) housekeeping genes were also selected for controls. Table 2: Primers and probes used in this study Gene Type Sequence Start Length (bp) Tm (°C) GC% Amplicon size (bp) PPP3CB Forward primer CCTCATGCCCTGGAATACAA (SEQ ID NO: 7) 2965 20 61.8 50 100 Reverse primer CCAACTTAGACAGGAGACCATAC (SEQ ID NO: 8) 3064 23 61.8 47.8 Probe TCCAGGCAGCGGAACCATCTATTG (SEQ ID NO: 9) TBL1XR1 Forward primer TCATCCTAAGTGCTGGAGTAGA (SEQ ID NO: 10) 1322 22 62.1 45.5 98 Reverse primer CAATGOTGGTGOTGAATGAAA (SEQ ID NO: 11) 1419 21 61.9 42.9 Probe TTGCTTGGCTTCACCAGTATGTGC (SEQ ID NO: 12) RNF213 (control) Forward primer GCTACGCATCCCTGCTATTT (SEQ ID NO: 13) 4571 20 62.3 50 103 Reverse primer AGGTACTGGTCCTTATCCAGAG (SEQ ID NO: 14) 4673 22 62.2 50 Probe AGCGTGGACTTCAGTGCATTCATGA (SEQ ID NO: 15) ACTG1 (control) Forward primer GTCACTTCGTGGCTAAGGTAAG (SEQ ID NO: 16) 1470 22 62.3 50 114 Reverse primer CTCAGCCCTGACACGTTAATAC (SEQ ID NO: 17) 1583 22 62.4 50 Probe ACACAGACTCACCAAGCCACAGAC (SEQ ID NO: 18) TRPM2 (1) Forward primer TCCGGCTCATGCACATTT (SEQ ID NO: 19) 2803 18 62.1 50 101101 Reverse primer CAGCAGGAAGAGGAAGAAGAAG (SEQ ID NO: 20) 2858 24 67.8 50 Probe TGTGAAGCGGATGATGAAGGACGT (SEQ ID NO: 21) 2903 22 62.2 50 TRPM2 (2) Forward primer ACAGCAACCACTCTCACTTC (SEQ ID NO: 22) 913 20 61.9 50 104 Reverse primer CTGCTCCGATATGAACTTCTCC (SEQ ID NO: 23) 1016 22 62.0 50 Probe TGGTCCTCAGAGGAATCTCCACC (SEQ ID NO: 24) TRPM2 (3) Forward primer CCTGTCTCGGACATCACTATCT (SEQ ID NO: 25) 1188 22 62.7 50 105 Reverse primer TTTGGTCCACTCGACAATCC (SEQ ID NO: 26) 1292 20 62.1 50 Probe TGATCCAGCAGAAACTGAGCGTGT (SEQ ID NO: 27) TRPM2 (4) Forward primer CTCTCTGGACTTCATCCTGTTC (SEQ ID NO: 28) 2777 22 61.7 50 102 Reverse primer TCCTTCATCATCCGCTTCAC (SEQ ID NO: 29) 2878 20 61.9 50 Probe AAATGTGOATGAGCCGGAGGC (SEQ ID NO: 30) TRPM2 (5) Forward primer GGAGGTGTACAAAGGCTACAT (SEQ ID NO: 31) 4346 21 61.7 47.6 109 Reverse primer GTTCAGCTCCACGTCATTCT (SEQ ID NO: 32) 4454 20 62.0 50 Probe ATGAOCCGAGGAACACGGACAATG (SEQ ID NO: 33) TRPM2 (6) Forward primer CTTCTGGTTTGGTGTCAACAG (SEQ ID NO: 34) 854 21 61,3 47,6 100 Reverse primer GCCTGAGATGGCAGATTTAATG (SEQ ID NO: 35) 953 22 61,5 45,5 Probe CT CACAGGAGCGT GAACCAT GAGG (SEQ ID NO: 36) TRPM2 (7) Forward primer AAGAGGGCCTCCATGTTTC (SEQ ID NO: 37) 577 19 61,7 52,6 101 Reverse primer CCCACGGAGTTTATCCTGAC (SEQ ID NO: 38) 677 20 61,7 55 Probe AGGTGCCTCAACATGGAGCCTT (SEQ ID NO: 39) TRPM2 (8) Forward primer CCACTCTATGCGAACCACAA (SEQ ID NO: 40) 61 20 62,1 50 142 Reverse primer CCAGGCTCAGGAGAGAGAA (SEQ ID NO: 41) 202 19 62,2 57,9 Probe CTGAGGGCACAGTCAGTAGTGAGC (SEQ ID NO: 42) TRPM2 (9) Forward primer CATCCACCATGGAGGTCATT (SEQ ID NO: 43) 469 20 61,8 50 139 Reverse primer TTGAGGCACCTCGAAACAT (SEQ ID NO: 44) 607 19 61,7 47,4 Probe CCCTCAGGCCTATGTCTGTGAGGA (SEQ ID NO: 45) TRPM2 (10) Forward primer AGGCGCATCCCACTCTAT (SEQ ID NO: 46) 52 18 62,6 55,6 86 Reverse primer TGAGGGCACAGTCAGTAGT (SEQ ID NO: 47) 137 19 62,3 52,6 Probe TTCTGGAGGAGGGTCTTGTGGTTC (SEQ ID NO: 48) MIR4323 Forward primer CCAGGCGGGCATGTGG (SEQ ID NO: 49) 7 16 65.2 75 60 Reverse primer CCCGAGGTCTGAGGCTGT (SEQ ID NO: 50) 66 18 65.1 66.7 Any nucleic acid or nucleotide sequence herein listed are shown using standard letter abbreviations for nucleotide bases and amino acids. In some cases, only one strand of each nucleic acid sequence is shown, while the complementary strand is understood as included by any reference to the displayed strand. A kit can be provided for performing the method described herein. The kit can include one or more primer sets for amplifying target regions of TBL1XR1, PPP3CB, TRPM2 or MIR4323, or any combination thereof. The primers may include one or more of the primers listed above or substantially similar primers (e.g. primers which have an 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity). The kit can also include one or more of the following: a reverse transcriptase, a DNA polymerase, a probe for each target gene (e.g. a probe listed above), buffer reagents, dNTPs, threshold or cut-off levels, instructions, in written or computer-implementable form, for performing the method described above. In the study described herein, the culturability of SARS-CoV-2 from size fractionated aerosols generated by ambulatory patients with COVID-19 during 2 follow-up visits was investigated (see Figure 1 for a study overview). In summary, between 25 and 60% of participants produced variant-specific culture positive aerosols <10 pm in diameter for up to 9 days after symptom onset, indicating considerable heterogeneity in the ability to produce culturable aerosols. Thus, only roughly a third of participants were probably highly infectious (produced culture-positive aerosols <5pm at -6 days after symptom onset) though we did not demonstrate person-to-person transmission. There was a relationship between nasopharyngeal PCR cycle threshold (Ct) value and aerosol culture positivity (including those probably highly infectious), and a 3 gene blood transcriptional signature was highly predictive of aerosol culture positivity. Although comorbidities and age did not predict aerosol culture positivity, the ability to produce culturable aerosols was associated with lower variant-specific neutralizing antibody titers and suppression of certain innate and adaptive components of host immunity (based on blood transcriptomic profiling). This study demonstrates the ability of COVID-19 patients to generate size-discriminated culturable virus in aerosol particles <10 pm, and relates aerosol culturability to clinical, viral and host determinants. The frequency of aerosol virus culturability was high at -60%. This is in contrast to published studies using size fractionated air sampling methodologies that have been mostly unsuccessful at culturing virus from aerosols generated from patients with acute SARS-CoV-2 infection. A gentle / low vacuum-based Andersen cascade impactor system (allowing graded multi-size discrimination) was used and (after various trial and error optimisation steps) agarose coated plates contained within the impactor were lightly sprayed with a thin film of viral culture medium, in tandem with rapid cold chain transportation to the laboratory to prevent viral dehydration. There was considerable heterogeneity in the ability of participants to generate viable virusladen aerosol and only -30% of participants were able to produce aerosols of <5 pm at visit 2. Furthermore, about a third of patients were probably non-infectious, 50% probably highly infectious, and -20% moderately infectious. These data may be in line with the ‘superspreader’ hypothesis, which assumes the presence of considerable heterogeneity in infectious probability with a limited proportion of persons being highly infectious driving a high proportion of transmission events. There is no consensus on what constitutes infectiousness, but it was reasoned that individuals who produced culturable aerosol for prolonged periods, in comparison to those who produced for limited periods, were more infectious. Almost a third of individuals were aerosol culture-positive for particles <10 pm despite having no respiratory symptoms (almost a quarter for particles <5 pm). Thus, consistent with other studies asymptomatic persons may be nasopharyngeal swab positive and infectious. However, this study shows for the first time that asymptomatic persons may also produce infectious aerosol <10 pm which is potentially suspensible for several hours and may be deeply inoculated by inhalation into the small airways and alveoli of the lung. Irrespective of particle size (0.65 pm to 7 pm) there was a clear relationship between increasing time from symptom onset and decreasing aerosol culturability, though -10% of individuals continued to emit infectious aerosol more than 8 days after symptom onset, again demonstrating considerable heterogeneity (discussed above). Consistent with prior work that evaluated NP culture positivity (but not aerosol positivity), this study found that nasopharyngeal Ct values correlated with the likelihood of infectiousness, and a Ct value <17 ruled in -40% of aerosol culture positive persons with a very high positive predictive value (almost 100%). A Ct value >27 effectively ruled out -25% of the tested population. Thus, in almost two-thirds of persons, nasopharyngeal Ct values (a readily available metric from PCR tests) could direct further clinical and public health interventions. It was also found that a simple 3-transcript blood-based biosignature was highly predictive of emitting culturable virus (PPV >90%). This readout could easily be ascertained using a multiplex PCR assay that could be automated within a point of care cartridge-based format (e.g., Xpert platform; Cepheid) to rapidly determine the likelihood of infectiousness. This might guide isolation periods and the need thereof, in certain professions with serious implications for transmission (e.g., health care and nursing home workers, airline workers, teachers, police officers etc.) or where there might be concern about exposing elderly or immunocompromised persons in the home setting. To the inventors’ knowledge this is the first proof-of-concept that a host transcriptomic signature can predict SARS-CoV-2 infectiousness. The 4 genes highly predictive of infectiousness were those encoding transcripts involved in RNA regulation (for example of CXCL10 a Th1 chemokine implicated in the COVID-19 hyperinflammatory syndrome or cytokine storm), transcription factor activation, and immune function suggesting that host pathogen relationships are important in the heterogeneity of infectiousness. The blood transcriptomic analysis revealed that several sets of pathways related to type 1 interferon, viral defence mechanisms, cytokine production / signalling, and adaptive immunity were predictably upregulated in those with culturable aerosol. However, specific innate immune pathways (those associated with complement and its activation) and pathways associated with suppression of humoral immunity (immunoglobulin production and B-cell immunity / receptor signalling) were also associated with aerosol culture positivity. This supported the study’s findings of low neutralising blood antibody levels being associated with increased aerosol culturability. Interestingly, it was found that pathways associated with nervous system processes, synaptic, and transsynaptic signalling were also relatively suppressed in those that were probably infectious compared to those that were probably non-infectious. This raises the possibility that neuroimmune modulation may impact the ability to produce aerosolised virus. It has recently been demonstrated that viral antigen can diffusely seed the central nervous system. Neuro-immuno-modulatory mechanisms can be effected through vagal efferents innervating the respiratory tract and this was associated with modulation of airway inflammation and immunity. This study’s data support the contention that host-pathogen interactions, including mucosal immunity, are critically important in determining host infectiousness. Thus, the higher demonstrated transmissibility of the Omicron variant in epidemiological studies may be related not to the ability to aerosolise virus, but due to other factors including differential host pathogen interactions including heterogeneity in receptor binding, NK cell responses, innate immunity, type 1 interferon responses, and adaptive immunity, the infecting dose, etc. In summary, the findings of this study indicated that SARS-CoV-2 is culturable from humangenerated aerosol <10 pm in almost 60% of the patients evaluated, although some individuals had the capability to aerosolise culturable virus in smaller particles for extended time periods. The ability to aerosolise culturable virus is heterogenous and inversely related to time from symptom onset and modulation of specific components of host immunity. Nasopharyngeal Ct thresholds and a preliminary unvalidated blood-based biosignature was highly predictive of infectious aerosol generation. These data support the need for prevention of airborne transmission risk using better ventilation in public transports, and indoor environments, especially hospitals, workplaces and schools, and the use of other airborne infection controls in health care facilities caring for COVID-19 patients. Potential host biosignatures have been identified that when coupled to positive diagnostic testing could serve as a proxy of probable infectiousness. As the biosignature can be determined in blood, this serves as a non-invasive and relatively quick, easy to perform method which does not require invasive and unpleasant sampling that is required by other methods (e.g. that require sputum or nasopharyngeal swabs). The invention will now be described in more detail by way of the following non-limiting examples. Examples: Methods Study design, eligibility criteria and regulatory approvals A diagnostic study, approved by the Human Research Ethics Committee at the University of Cape Town (HREC REF: 387 / 2020), was used to identify and recruit participants into this study. Informed consent was obtained from all participants. A cross-sectional study design with prospective follow-up was used. Eligibility criteria included being >18 years, testing positive for COVID-19 by PCR or rapid antigen, ambulatory, being within 7 days of symptom onset, having a blood oxygen saturation (O2 SAT) <95 and willing to undergo CASS and provide nasopharyngeal and saliva specimens. All laboratory aspects of this study were approved by the University of Cape Town Institutional Biosafety Committee prior to study initiation. SARS-CoV-2 culture was performed in a BSL-3 laboratory by vaccinated personnel utilizing powered air purifying respirators. Patient clinical information, sample collection and CASS procedure From January 2021 to May 2022, 44 ambulatory patients with PCR-confirmed COVID-19 who underwent CASS were recruited (designated visit 1; Figure 1A). A second CASS sampling was undertaken ~48 to 72 hours after visit 1 (designated visit 2; 38 participants completed this visit; Figure 1A). Staff used N95 masks and wore comprehensive personal protective eguipment. Consenting patients underwent a nurse-administered clinical and sociodemographic interview. Sex of every participant was captured as part of this interview and was a self-reported status. Peak expiratory flow (PEF), forced expiratory volume (FEV) and cough peak flow (CPF) were measured using an Asma-1 Electronic Respiratory Monitor (Vitalograph, United Kingdom) and a Respi-Aide Peak Flow Meter (GaleMed, China). The average of three consecutive blows were used. All procedures were done at one visit in the same order. A nasopharyngeal swab in viral transport medium (VTM), saliva samples and blood for host immunity (Figure 1C) interrogation were collected prior to aerosol sampling. Viral transport medium (VTM) was prepared in the laboratory using Anderson’s modified Hanks Balanced Salt Solution (8.0 g / l NaCI, 0.4 g / l KCI, 0.05 g / l Na2HPO4, 0.06 g / l KH2PO4, 1.0 g / l Glucose, 0.7 g / l NaHCOs, 0.2 g / l MgSO4.7H2O, 0.14 g / l CaCI2.2H2O) with 2% v / v heat-inactivated fetal calf serum, 100 pg / ml gentamicin, 100 I.U / ml penicillin, 100 pg / ml streptomycin and 2.5 pg / ml of Amphotericin B. CASS was performed in a negative pressure well-ventilated sampling cubicle embedded within a larger negative pressure room. The CASS apparatus consisted of a facemask attached to tubing connected to a vacuum-based Anderson cascade impactor (see Figure 1B) within a metal drum, with the flow rate monitored using a flow meter and kept at around 30 L / min. Aerosol sampling was conducted using viral medium coated agar plates located at 6 stages (collects aerosol in a particle size-dependent manner) within the Anderson impactor, a settle plate at head level, placed 50 cm away from participant within the cubicle. Briefly, patients coughed as forcefully and as frequently as possible into the CASS for 5 min (5 sequences of 1 min cough followed by 1 min rest) via a 1 m silicone pipe that ran from the patient in a sputum induction booth into the CASS. Ambient temperature and humidity and the number of coughs were recorded. CASS and sample processing The CASS was transported back to the laboratory under cold condition (maintained using ice packs) as soon as sampling was completed. Longest time between end of sampling and delivery of system was 45 minutes. The impactor was removed from its encasing drum in a biological safety cabinet within the BSL3 facility. Plates were sequentially removed, irrigated each with 1.2 ml of cell culture medium, with the plates carefully and gently swirled to ensure irrigated medium swept across the full area of the agar layer. Around 900-1000 pl of media was recovered and frozen down into 3 separate aliquots fordownstream PCR and viral culture. Settle plate was treated in a similar way. Nasopharyngeal swab was vortexed for 5 sec and VTM aliquoted and stored at -80°C. 1.2 ml of cell culture media was added to the saliva specimen, mixed, aliquoted, and stored at-80°C. PAXgene blood was left at room temperature overnight and frozen at -80°C until RNA extraction. Serum was collected after centrifugation at 500x g for 10 minutes, aliquoted and stored at -80°C until neutralization assay. Polymerase chain reaction of raw samples and viral culture supernatant Nucleic acid amplification test using the Emergency use authorization assay (Catalog # 2019-nCoVEUA-01) developed by the USA Centers for Diseases Control and Prevention (CDC) was used to detect SARS-CoV-2 in respiratory samples and viral culture supernatant. The panel was designed for specific detection of SARS-CoV-2 (two primer / probe sets targeting the nucleocapsid gene). An additional primer / probe set to detect the human RNase P gene (RP) in control samples and clinical specimens was also included in the panel. Results were classified as positive for SARS-CoV-2 when both the N1 and N2 targets of the nucleocapsid gene were detected by PCR with cycle threshold (Ct) values were <40. Viral culture To establish the in vitro viral culture model, a SARS-CoV-2 viral stock (isolated from a COVID-19 patient during the Beta wave) was used to infect the human lung carcinoma cell line, H1299 (ATCC CRL-5803), in a BSL3 laboratory and infection was confirmed by light microscopy (as assessed by cytopathic effects of the virus on the cell line) and confocal microscopy (Figure 1D). Serial dilutions of the viral stock were used to establish the limit of detection of the PCR assay at 1x101 copies / ml. Virus isolation culture was attempted from the nasopharyngeal swab, saliva, cough tubing, settle plates and CASS plates. The cell line was maintained in Roswell Parks Memorial medium (RPMI) containing 10% bovine serum, 100 III penicillin / streptomycin, 2 Mm L-glutamine, 25 Mm HEPES, 1x non-essential amino acids and 0.1 mg / ml sodium pyruvate (ThermoFisher, South Africa). All sample aliquots destined for viral culture were initially filtered through a 0.22 pm filter prior to inoculation. The samples were diluted 2-folds using cell culture media during the filtration step. 250 pl of filtered samples were inoculated in respective wells and cultures were grown in a humidified 37°C incubator with 5% CO2 and cytopathic effect (CPE) (Figure 1D) and viral replication (Figure 1E) were monitored on days 1, 3, 6 and 9 by PCR. Viral culture positivity was defined as at least a 100-fold increase in viral load over time. Reproducibility of the viral culture assay was ascertained through the duplicate culture of a subset of samples (nasopharyngeal swab and CASS plates), in separate culture experiments. Replicability was defined as similar qualitative outcomes, that is, culture positive or negative in both runs. SARS-CoV-2 whole genome sequencing and genome assembly Viral sequencing was performed through the Network for Genomics Surveillance in South Africa (NGS-SA), at the University of Cape Town Division of Medical Virology. RNA was extracted from harvested viral-culture supernatant (Vero E6 or H1299 cells) on an automated MaelstromTM 4800 using the TANBead® Nucleic Acid Extraction Kit (Taiwan Advanced Nanotech Inc, Taipei, Taiwan) or ChemagicTM 360 automated system (PerkinElmer, Inc, Waltham, MA) as per manufacturer’s protocol. Whole genome amplification and library preparation were performed using the Illumina COVIDSeq Test kit and protocol 1000000128490 v02 (Illumina, Inc., San Diego, CA), and executed on the Hamilton Next Generation Starlet (Hamilton Company, Reno, NV). Whole genome amplification was achieved via multiplex polymerase chain reaction performed with the ARTIC V4.1 primers designed to generate 400-bp amplicons with an overlap of 70 bp that spans the 30 kb genome of SARS-CoV-2. Indexed paired-end libraries were normalized to 4 Nm concentration, pooled, and denatured with 0.2 N sodium acetate. A 4Pm pooled library was spiked with 1% PhiX Control v.3 adaptor-ligated library (Illumina, Inc., San Diego, CA) and sequenced using the MiSeq® Reagent Kit v2 (500 cycle) and sequenced on the MiSeq instrument (Illumina, Inc., San Diego, CA). The quality of sequencing reads was assessed using different tools including FastQC, Fastp, Fastv, Fastq_screen, and Fastx_toolkit. The resulting reads were analyzed on Exatype (exatype.com) for referenced-based genome assembly and to identify minor and major variants. The assembled consensus sequences were analyzed using Nextclade Web (www.nextstrain.org) for further quality control and clade assignment. The Stanford Coronavirus Resistance Database (CoV-RDB; www.covdb.stanford.edu), designed to house comprehensively curated published data on the neutralizing susceptibility of SARS-CoV-2 variants and spike mutations to monoclonal antibodies, convalescent plasma, and vaccinee plasma, was used in mutational analysis to identify relevant mutations in the sequenced viruses and allow for comparative assessment of their occurrence across different phenotypes of participants. Neutralization assay The 293T / ACE2.MF cells modified to overexpress human ACE2 were provided by Dr Mike Farzan, Scripps Research. These cells were cultured in DMEM containing 10% heat-inactivated fetal calf serum and 3 pg / ml puromycin at 37 °C, 5% CO2. Cell monolayers were disrupted at confluency by treatment with 0.25% trypsin in 1 Mm EDTA. SARS-CoV-2 pseudotyped lentiviruses were prepared by co-transfecting the HEK293T cell line with either the SARS-CoV-2 Beta spike (L18F, D80A, D215G, K417N, E484K, N501Y, D614G, A701V ,242-244del) or the Delta spike (T19R, R158G L452R, T478K, D614G, P681R, D950N, 156-157 del) plasmids in conjunction with a firefly luciferase encoding lentivirus backbone plasmid. For the neutralization assay, heat-inactivated plasma samples from participants were incubated with the SARS-CoV-2 pseudotyped virus for 1h at 37C, 5% CO2. Choice of pseudotyped lentivirus system used (Beta or Delta spike) was based on autologous hostvariant pairing, that is, participants infected with the Beta variant had their serum tested with the Beta spike, and likewise, participants infected with the Delta variant had their serum tested with the Delta spike. Subsequently, 1x104 HEK293T cells engineered to overexpress ACE-2 were added and incubated at 37C, 5% CO2 for 72 h upon which the luminescence of the luciferase gene was measured. CB6 was used as a positive control. Neutralization was measured as described by a reduction in luciferase gene expression after single-round infection of 293T / ACE2.MF cells with spike-pseudotyped viruses. Titers were calculated as the reciprocal plasma dilution (ID50) causing 50% reduction of relative light units. All assays were run in duplicate. Blood host transcriptomics Blood RNA sequencing was performed on 33 individuals (21 CASS-positive and 12 CASS-negative) using blood collected over a 48-hour period (visit 1 and 2; figure 1A). In brief, total RNA was extracted from PAXgene blood RNA tubes using the PAXgene blood RNA isolation kit (Qiagen, PreAnalytix; catalog: 762174). Sequencing libraries were prepared using RNA and globin depletion and sequenced on the Illumina platform using 150bp pair-end sequencing paired-end reads. The FastQC program (version 0.11.9, www.bioinformatics.babraham.ac.uk / projects / fastqc / ), was used to assess read quality and trimming was performed using the Trim Galore program (version 0.6.10,.bioinformatics.babraham.ac.uk / projects / trim_galore / ). The Spliced Transcripts Alignment to a Reference (STAR) software (version STAR_2.7.7a) (PMID: 23104886) was used to map reads to the Ensembl (PMID: 33137190) human genome primary assembly (version GRCh38.99) with the quantMode and GeneCounts option selected to generate raw genewise read counts for each sample. The DE analysis was performed with the edgeR (version 3.38.4) (PMID: 19910308) Bioconductor package. Briefly, raw counts were filtered to remove genes with low expression, normalized, and negative binomial generalized linear models were fitted. The quasi-likelihood F-test was used to identify DE genes when comparing CASS-positive to CASS-negative individuals. A gene set enrichment analysis (GSEA) for Gene Ontology (Biological Process) was performed on the differential expression results ranked by fold change using the gseGO function, from the clusterProfiler (ver: .4.4.4, PMID: 34557778) package in R. The simplify function was used to reduce redundancy in the enriched pathways. Predictive Modeling Predictive modeling was performed on the transcriptional data using the random forest ranger algorithm in the tidymodels program (version, 1.0.0) in R. The 5 most important variables were identified from the 100 genes with the smallest p-values from the differential expression analysis. Predictive modelling was subsequently performed with 1000 bootstraps using various combinations of the top 5 most important variables and the best models were selected that used 2 to 3 gene biomarkers. Statistical analysis The Fisher’s exact test was used for comparisons involving categorical outcomes, Mann-Whitney test was used for non-parametrically distributed continuous data, and logistic regression performed to generate odds ratios (Stata version 17 or GraphPad, Version 9.4.1). A p-value of <0.05 was considered significant for all statistical analyses, all tests were two-tailed and adjustment for multiple comparisons was limited to the transcriptomic analysis. Receiver operating characteristic (ROC) curves were generated for sensitivity / specificity analysis. The DE results were sorted / ranked by fold change and a gene set enrichment analysis (GSEA) for Gene Ontology (Biological Process) and KEGG pathways was performed using the gseGO and gseKEGG functions respectively, from the clusterProfiler (ver: .4.4.4 package in R. Pathways with an FDR <0.05 were considered significant. Primer design The PrimerQuest™ Tool (Integrated DNA Technologies - eu.idtdna.com / paqes / tools / primerquest) a program of choice for designing qPCR primers / probes, sequencing oligonucleotides, and custom primers, was used to design the current set of primers reported herein. Designed primers were subjected to BLAST (blastn) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and the specific candidates were used for analytical sensitivity and specificity testing. Amongst other things, the designed primers were assessed for their ability to form dimers. Polymerase chain reaction (PCR) The technique for measuring mRNA is reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). Here, a DNA template is made from mRNA using reverse transcription. This template, which is called cDNA (complimentary DNA) is then amplified. As the DNA amplification proceeds, hybridization probes emit changing levels of fluorescence, which can be used to measure the original number of mRNA copies. Tables 3 and 4 show the PCR mix and PCR conditions. Table 3: PCR MasterMix Composition Component Concentration Buffer 1X MgCI2 3 mM dNTPs 0.8 mM Fwd Primer 200 nM Rev Primer 200 nM Taq 1.25 U MilliQ N / A DNA N / A Table 4: Optimised PCR Cycling Parameters Temperature Time No. of cycles Initial Denaturation 95°C 3 min N / A Denaturation 95°C 30 sec 30 cycles Annealing 57°C 30 sec Elongation 72°C 30 sec Final Elongation 72°C 5 min N / A 5 Results Study design and description of cases A total of 44 participants were enrolled and 38 of them completed a second visit for CASS 10 within 2-3 days after the first procedure. There was an almost equal split across sex (21 males and 23 females), average age of participants was 38 years, ten (22%) reported a smoking history, 13 (30%) had received at least one dose of COVID-19 vaccine, and 11 (25%) reported having a risk factor other than a BMI >25 (n=22; 50%). Forty (91%) participants reported being symptomatic and 38 (86%) reported at least one respiratory symptom (cough, sore throat, 15 shortness of breath or chest pain). Whole genome sequencing for viral variant classification (or date of infection) confirmed ten participants with Beta, 27 with Delta and 7 with Omicron. Table 5 provides a full breakdown of participants’ characteristics. Table 5: Demographic and clinical characteristics of aerosol culture positive (10pm and 5pm) versus aerosol culture negative ambulatory persons. n (total visits = 82) All participants N= 44 Culture negative aerosol N= 36' Culture positive aerosol (<10 pm) N= 46 t Culture positive aerosol (<5 pm) N= 33 • Sex (%) Male Female 21 (48) 23 (52) 19(53) 17(47) 20 (43) 26 (57) 14 (42) 19 (58) Average age / years (range) 38 (20-71) 37 (20-59) 38 (21-71) 39 (21-71) BMI (%) Normal Overweight Obese 22 (50) 15 (34) 7 (16) 16(44) 13(36) 7(20) 24 (52) 16(35) 6 (13) 16 (48) 13 (39) 4(12) Risk Factor (%) None Hypertension Asthma Diabetes HIV Anemia Pregnancy 33 (75) 5(11) 3(7) 1 (2) 1 (2) 1 (2) 1 (2) 30 (83) 3(8) 0(0) 0(0) 0(0) 2(6) 1 (3) 33 (72) 5(11) 6(13) 1 (2) 1 (2) 0(0) 1 (2) 22 (67) 5(15) 5(15) 1 (3) 1 (3) 0(0) 0(0) Current Smoker (%) 10(22) 9 (25) 9(20) 5(15) Vaccinated (%) 13(30) 12(33) 11 (24) 4(12) Symptomatic Any (%) Median Duration / days (IQR) Respiratory (%) Median Duration / days (IQR) Non-respiratory (%) Median Duration / days (IQR) 40 (91) 4 (3-6) 38 (86) 3 (3-6) 33 (75) 4 (3-6) 28 (78) 6 (4-9) 23 (64) 6 (3-8) 24 (67) 5 (3-8) 44 (96) 5 (3-6) 41 (89) 5 (3-6) 35 (76) 5 (3-6) 32 (97) 5 (3-6) 29 (88) 5 (3-6) 25 (76) 5 (3-6) Cough metrics Median number of coughs (IQR)* Median peak flow (IQR) Median peak cough flow (IQR) 182 (140-231) 318 (250-408) 380 (263- 487) 186 (154-231) 347 (262-443) 398 (304-500) 177 (124-234) 310 (216-395) 360 (239-487) 186 (119-244) 300 (208-408) 374 (263-501) t visits 1 and 2 were aggregated. * gentle cough over a 10-minute period (coughing occurred for a period of 1 min with alternating 5 periods of 1 min of rest i.e. total cough duration was 5 minutes). No significant differences between tabulated variables were observed between the three groups. All the participants had an oxygen saturation over> 95% at sea level. Frequency of aerosol culture positivity First, the frequency and duration of culture positive aerosol generation was investigated. It was found that between 50% (visit 2; a median of 6 days after symptom onset) to 60% (visit 1; a median of 4 days after symptom onset) of participants (n=44) generated aerosols of <10 pm (0.65 to 7 pm particle median diameter), and between 30% (visit 2) to 50% (visit 1) produced aerosols <5 pm (0.65 to 4.7 pm) (see Figure 2A that outlines the numbers and proportion of patients by visit). Thus, of the 44 participants, 13 were aerosol culture negative and 31 were aerosol culture positive (n= 15 at both visits; n= 7 at visit 1 but not visit 2; n= 4 at visit 2 but not visit 1; n=5 at visit 1 but did not complete visit 2). Notably, virus was able to be cultured from only 10% of viral medium impregnated settle plates that were located within 50 cm (0.5 m) from the patient. The complete size fragmentation of sampled aerosols (across 6 median particle sizes) for the two CASS visits is displayed in Figure 2B. Heterogeneity of aerosol culture positivity There was considerable heterogeneity in the ability of participants to generate detectable aerosols and only 29% of participants produced aerosols of <5 pm at visit 2 (at a median of 6 days after symptom onset), thus broadly in line with the heterogeneity of infectiousness as seen with other respiratory infections such as measles, influenza, and tuberculosis. The variability outlined could not be explained by clinical or demographic variables (Table 5). The relationship between probably highly infectious persons and patient and viral characteristics was evaluated (Table 6). Nasopharyngeal Ct value was negatively associated with probably highly infectious persons (defined as being aerosol positive on visit 2, i.e., median of 6 days after symptom onset in contradistinction to moderately infectious persons [only aerosol positive on visit 1] and probably non-infectious [aerosol culture negative]). Three participants were initially tested with exhaled breath and no coughing, and subsequently completed the cough protocol. Two of the three were cough aerosol culture positive but all three were aerosol culture negative using the exhaled breath and no coughing protocol, which was abandoned in the early phase of the study (as performing both protocols was not pragmatic from a workload or cost point of view). Table 6: Heterogeneity in aerosol culture positivity (probable infectiousness) in those for whom aerosol culture data was present for both visits (n= 38). Probably highly infectious Probably non-infectious (culture -ve) OR (95% Cl)* p-value Probably moderately infectious OR (95% Cl) vs probably highly infectious0 p-value OR (95% Cl) vs probably non-infectious* p-value n (% of total) 19(50) 12(32) 7(18) Median NPS Ct (IQR) t Culture 16.9 (14.5-21.8) 24.1 (19.0-34.9) 1.2(1.0-1.4) 0.01 19.1 (11.3-20.8) 1.0(0.7-1.2) 0.93 1.2(1.0-1.5) 0.12 positive NPS (%)* 18 (95) 7(58) 12.9(1.3-131) 0.02 5(71) 7.2 (0.6-96.7) 0.17 1.8 (0.2-13.2) 0.66 Median cough tubing Ct (IQR)t 20.5 (17.4-24.9) 26.8 (24.0-37.9) 1.2 (1-1.3) 0.003 23.3 (22.9-30.1) 1.2 (1.0-1.5) 0.06 1.0 (0.7-1.2) 0.35 Culture positive cough tubing (%) t 16(84) 4(33) 10.7(1.9-59.4) 0.007 5(71) 2.1 (0.3-16.6) 0.59 5.0 (0.7-38.2) 0.17 Respiratory symptoms at visit (%) t Median duration 15 (79) 7(58) 2.7(0.5-13.2) 0.25 5(71) 1.5 (0.2-10.8) 0.65 1.8 (0.2-13.2) 0.66 respiratory symptom / days 3 (3-4.3) 6(2.5-6) 1.4(0.9-2.2) 0.27 3 ¢2.5-5) 1.1 (0.5-2.8) 1 1.0 (0.5-2.2) 0.38 (IQR) t Median number of coughs (IQR) t 169(121-235) 186(158-229) 1.0(1.0-1.0) 0.48 208(163-235) 1.0(1.0-1.0) 0.33 1.0(0.9-1.0) 0.64 Sex (%) Male 7(37) 7(58) 0.4(0.1-1.8) 0.29 4(57) 0.4 (0.1-2.6) 0.41 1.0 (0.1-6.3) 1.00 Female 12 (63) 5(42) 3(43) Median Age / years (IQR) 36 (29-42) 36 (27-50) 0.98 33 (27-40) 0.58 0.67 BMI (%) Normal 9(50) 6(50) 3 (43) Overweight 7(39) 3(25) 0.6(0.1-3.5) 0.69 4(57) 1.7(0.3-10.3) 0.67 0.4 (0.05-2.9) 0.61 Obese 3(11) 3(25) 1.5(0.2-10.1) 1.00 0(0) 1.0(0.07-13.7) 1.00 1.5(0.11-21.3) 1.00 Risk Factor (%) Yes No 5(26) 14 (74) 2(17) 10 (83) 1.8(0.3-11.1) 0.68 1 (14) 6 (86) 2.1 (0.2-22.5) 0.65 0.8 (0.06-11.3) 1.00 Vaccinated (%) 4(27) 4(33) 2 (29) Viral (%) Delta Beta Omicron 13(68) 5(26) 1(6) 5(42) 4 (33) 3(25) 2.1 (0.4-11.1) 7.8 (0.6-93.9) 0.42 0.12 6 (86) 0(0) 1 (14) 0.4 (0.04-4.6) 2.2(0.1-40.8) 0.64 1.00 4.8(0.4-58.1) 3.6 (0.3-46.4) 0.31 0.57 Non-infectious (Nl) = CASS negative at both visits (median of D3 and 6 after symptom onset; n=12); probably highly infectious = cases positive at visit 2 (n=19); probably moderately infectious = CASS positive at one visit 1 but not visit 2 (n= 7). Note: n=6 only attended visit 1. For the purposes of calculating median Ct value only visit 1 was used as this best reflected Ct at or close to diagnosis. The Fisher’s exact test was used for comparisons involving categorical outcomes, Mann-Whitney test was used for non-parametrically distributed continuous data. * Probably highly infectious as reference group 0 probably highly infectious as reference group t probably moderately infectious as reference group Relationship between aerosol positivity and symptoms and viral genetic variants Although respiratory symptoms (cough, sore throat, shortness of breath, chest pain) were associated with infectiousness (Figure 2C), almost one-third of aerosol culture positive individuals had no respiratory symptoms or were asymptomatic, making this metric unreliable for guiding public health interventions. Itwas further shown that the likelihood of infectiousness was highest within the first 8 days of symptom onset (Figure 2D) in keeping with human lung challenge and other studies that conducted viral culture of samples from the upper respiratory tract19. It could not be definitively ascertained whether any specific viral variant (Beta, Delta versus Omicron) was associated with greater infectiousness (Figure 3A), and no evidence was found that viral genomic variants including mutations in the envelope-encoding proteins (potentially portending ability to better withstand desiccation and UV light) could explain infectiousness. Relationship between aerosol positivity and host immunity (neutralising antibodies and blood mRNA seguencing) Next, host immunity and its relationship to infectiousness was studied. It was found that the ability to aerosolise culturable virus was significantly associated with low variant-specific serum neutralizing antibody levels (Figures 3B &3C). The blood mRNA sequencing-related differential expression (DE) analysis identified a total of 11 upregulated (TBL1XR1, PPP3CB, TASOR, ZHX1, ARL8B, CWC22, TNPO1, CDC5L, CNOT8, SMC3, RIMOC1, KPNA3, ABRAXAS2) and 9 downregulated (DNASE1L2, TRPM2, UBAP1L VASN, ABCA3, BRSK2, SEMA6C, MIR4323, FTCD) genes when comparing CASS-positive to CASS-negative individuals (FDR <0.05). By contrast, at an uncorrected p-value cut-off of <0.05, the same comparison identified 1735 upregulated and 1278 downregulated genes. The gene set enrichment analysis (GSEA) of the related gene ontology biological process pathways, identified significant enrichments (p-value <0.05) in activated and suppressed pathways when comparing CASS-positive to CASS-negative individuals. Pathways significantly activated in CASS-positive individuals include responses to biotic stimuli including virus and bacteria, innate and inflammatory immune responses as well as production and responses to cytokines and type I interferons. Pathways suppressed in CASS positive individuals, included those related to complement and B cell activation, phagocytosis, and immunoglobulin mediated immune responses, and those related to ion transport, development, and neuronal sensory perception Diagnostic predictors of infectiousness (nasopharyngeal swab Ct value and blood-based biosignature) A number of biological patterns predicted infectiousness. Firstly, it was found that a Ct value of <16.3 (using the CDC 2019-Novel Coronavirus (2019-nCoV) RealTime Reverse Transcriptase (RT)-PCR Diagnostic Kit) ruled in ~40% of aerosol culture-positive participants (i.e., modest sensitivity but very high specificity and thus high confidence that they were infectious; Figures 3D &3E). The exclusion of participants above a Ct value of greater than 27 (negative predictive value) was less helpful in itself as it ruled out -20% of the tested population as being highly likely to be non-infectious (i.e., high confidence that they were non-infectious). However, a combination of both could be useful to determine public health strategy in almost two thirds of persons (high confidence infectious and non-infectious persons). When time from symptom onset was combined with Ct value, rule-in value only marginally increased to 42.5%. A strong association was observed between the capacity to produce culture positive aerosols and having either a culture positive nasopharyngeal swab, culture positive saliva or culture positive cough tubing sample. Accuracy of nasopharyngeal swab Ct as a proxy for the identification of selected potentially highly infectious cases is summarised in Table 7 (and the ROC from the data plotted in Figure 4). Table 7: Accuracy of nasopharyngeal swab Ct as a proxy for identification of highly infectious cases Sensitivity (95% Cl) Specificity (95% Cl) Positive Predictive Value (95% Cl) Negative Predictive Value (95% Cl) Accuracy (95% Cl) Ct cut-off 94.74 (74-100) 58.33 (28-85) 78.26 (56-93) 87.50 (47-100) 80.65 (63-93) 22.6 89.47 (28-85) 58.33 (28-85) 77.27 (55-92) 77.78 (40-97) 77.42 (59-90) 22.4 78.95 (54-94) 58.33 (28-85) 75 (51-91) 63.64 (31-89) 70.97 (52-86) 21.9 68.42 (43-87) 58.33 (28-85) 72.22 (47-90) 53.85 (25-81) 64.52 (45-81) 19.5 57.89 (34-80) 83.33 (52-98) 84.62 (55-98) 55.56 (31-78) 67.74 (49-83) 17.1 47.37 (24-71) 83.33 (52-98) 81.82 (48-98) 50.00 (27-73) 61.29 (42-78) 16.9 42.11 (20-67) 91.67 (62-100) 88.89(52-100) 50.00 (28-72) 61.29 (42-78) 16.6 31.58 (13-57) 91.67 (62-100) 85.71 (42-100) 45.83 (26-67) 54.84 (36-73) 14.6 21.05 (6-46) 91.67 (62-100) 80.00(28-100) 42.31 (23-63) 48.39 (30-67) 13.8 10.53 (1-33) 91.67 (62-100) 66.67 (9-99) 39.29 (22-59) 41.94 (25-61) 13.6 Identification of gene biosignatures for infectiousness Predictive modelling using the transcriptional data identified a number of models that achieved high specificity and sensitivity using 2 and 3 gene blood biomarker combinations. Models that achieved a sensitivity and specificity >90% included the 3 biomarker combinations of MIR4323, TBL1XR1, PPP3CB and MIR4323, TBL1XR1, TRPM2 as did the 2 gene combination of TBL1XR1 and PPP3CB (Figure 5). The combination of PPP3CB, TBL1XR1 and TRPM2 was identified as being particularly promising (Table 8). A few other 2 biomarker combinations and single gene biomarkers achieved sensitivities >90% and specificities >80%. Table 8: Modelling information for the gene combination of PPP3CB, TBL1XR1 and TRPM2 for predicting infectiousness as measured by capacity of patients to emit culture positive aerosols. PPP3CB, TBL1XR1, TRPM2 Probability cut-off 0.5 Sensitivity 0.95 (0.76, 1) Specificity 0.82 (0.48, 0.98) PPV 0.91 (0.71,0.99) NPV 0.9 (0.55, 1) AUC 0.97 PPV: Positive predicting value; NPV: Negative predictive value; AUC: Area under curve.; TBL1XR1: transducing (beta)-like 1X-linked WD40 repeat-containing gene, PPP3CB: protein phosphatase 3 catalytic subunit beta; TRPM2: transient receptor potential cation channel, subfamily M, member 2. An F-test was used to compare CASS-positive to CASS-negative participants. PCR The primers and probes of Table 2 were shown to specifically target the genes of interest. The foregoing description has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure. The language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims. Finally, throughout the specification and accompanying claims, unless the context requires otherwise, the word ‘comprise’ or variations such as ‘comprises’ or ‘comprising’ will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claims

1. A method of predicting COVID-19 infectiousness, the method comprising: determining expression of the TBL1XR1 gene in a biological sample from a subject having or possibly having COVID-19;optionally also detecting expression of at least one of the PPP3CB, TRPM2 and MIR4323 genes;wherein the level of expression of TBL1XR1 or at least one of the other genes indicates the likely infectiousness of the subject.

2. The method of claim 1, wherein the expression ofTBL1XR1 and PPP3CB is determined.

3. The method of claim 1, wherein the expression of TBL1XR1 and Ml R4323 is determined.

4. The method of claim 1, wherein the expression of TBL1XR1 and TRPM2 is determined.

5. The method of any one of claims 1,2 or 4, wherein the expression of TBL1XR1, PPP3CBand TRPM2 is determined.

6. The method of any one of claims 1 to 3, wherein the expression of TBL1XR1, PPP3CB and MIR4323 is determined.

7. The method of any one of claims 1, 3 and 4, wherein the expression of TBL1XR1, MIR4323 and TRPM2 is determined.

8. The method of any one of claims 1 to 7, wherein the sample is a blood sample.

9. The method of any one of claims 1 to 8, wherein the sample is from a subject who hastested positive for COVID-19.

10. The method of any one of claims 1 to 9, wherein the expression of the gene(s) is determined by determining the mRNA level of the gene(s) in the sample.

11. The method of any one of claims 1 to 10, wherein the expression of the gene(s) is determined by performing PCR.

12. The method of any one of claims 1 to 11, wherein the gene expression in the sample is compared with a reference level of the same gene(s) in order to determine whether the subject is infectious.

13. A primer pair for use in a method of amplifying the TBL1XR1 gene, the primer pair comprising:Forward primer: TCATCCTAAGTGCTGGAGTAGA (SEQ ID NO: 10); andReverse primer: CAATGCTGGTGCTGAATGAAA (SEQ ID NO: 11).

14. A primer pair for use in a method of amplifying the PPP3CB gene, the primer pair comprising:Forward primer: CCTCATGCCCTGGAATACAA (SEQ ID NO: 7); andReverse primer: CCAACTTAGACAGGAGACCATAC (SEQ ID NO: 8).

15. A primer pair for use in a method of amplifying the TRPM2 gene, the primer pair selected from the group consisting of:a) Forward primer: TCCGGCTCATGCACATTT (SEQ ID NO: 19); and Reverse primer: CAGCAGGAAGAGGAAGAAGAAG (SEQ ID NO: 20);b) Forward primer: ACAGCAACCACTCTCACTTC (SEQ ID NO: 22); and Reverse primer: CTGCTCCGATATGAACTTCTCC (SEQ ID NO: 23);c) Forward primer: CCTGTCTCGGACATCACTATCT (SEQ ID NO: 25); and Reverse primer: TTTGGTCCACTCGACAATCC (SEQ ID NO: 26);d) Forward primer: CTCTCTGGACTTCATCCTGTTC (SEQ ID NO: 28); and Reverse primer: TCCTTCATCATCCGCTTCAC (SEQ ID NO: 29);e) Forward primer: GGAGGTGTACAAAGGCTACAT (SEQ ID NO: 31); and Reverse primer: GTTCAGCTCCACGTCATTCT (SEQ ID NO: 32);f) Forward primer: CTTCTGGTTTGGTGTCAACAG (SEQ ID NO: 34); and Reverse primer: GCCTGAGATGGCAGATTTAATG (SEQ ID NO: 35);g) Forward primer: AAGAGGGCCTCCATGTTTC (SEQ ID NO: 37); and Reverse primer: CCCACGGAGTTTATCCTGAC (SEQ ID NO: 38);h) Forward primer: CCACTCTATGCGAACCACAA (SEQ ID NO: 40); and Reverse primer: CCAGGCTCAGGAGAGAGAA (SEQ ID NO: 41);j) Forward primer: AGGCGCATCCCACTCTAT (SEQ ID NO: 46); and Reverse primer: TGAGGGCACAGTCAGTAGT (SEQ ID NO: 47).

16. A kit for predicting COVID-19 infectiousness, comprising:a) a primer pair for amplifying TBL1XR1; andb) a probe for detecting the TBL1XR1 gene.

17. The kit of claim 16, further comprising at least one primer pair and probe for amplifying and detecting PPP3CB, TRPM2 and / or MIR4323.

18. The kit of claim 16 or 17, wherein:the nucleotide sequences of the primer pair for amplifying TBL1XR1 are:Forward primer: CCTCATGCCCTGGAATACAA (SEQ ID NO: 10); and Reverse primer: CCAACTTAGACAGGAGACCATAC (SEQ ID NO: 11); and / orthe nucleotide sequence of the probe for detecting the TBL1XR1 gene is TTGCTTGGCTTCACCAGTATGTGC (SEQ ID NO: 12).

19. The kit of claim 17 or 18, wherein:the nucleotide sequences of the primer pair for amplifying PPP3CB are:Forward primer: CCTCATGCCCTGGAATACAA (SEQ ID NO: 7); and Reverse primer: CCAACTTAGACAGGAGACCATAC (SEQ ID NO: 8); and / orthe nucleotide sequence of the probe for detecting the PPP3CB gene is TCCAGGCAGCGGAACCATCTATTG (SEQ ID NO: 9).

20. The kitof any one of claims 17 to 19, wherein:the nucleotide sequences of the primer pair for amplifying TRPM2 are selected from the group consisting of:a) Forward primer: TCCGGCTCATGCACATTT (SEQ ID NO: 19); and Reverse primer: CAGCAGGAAGAGGAAGAAGAAG (SEQ ID NO: 20);b) Forward primer: ACAGCAACCACTCTCACTTC (SEQ ID NO: 22); and Reverse primer: CTGCTCCGATATGAACTTCTCC (SEQ ID NO: 23);Reverse primer: TTTGGTCCACTCGACAATCC (SEQ ID NO: 26);d) Forward primer: CTCTCTGGACTTCATCCTGTTC (SEQ ID NO: 28); and Reverse primer: TCCTTCATCATCCGCTTCAC (SEQ ID NO: 29);e) Forward primer: GGAGGTGTACAAAGGCTACAT (SEQ ID NO: 31); and Reverse primer: GTTCAGCTCCACGTCATTCT (SEQ ID NO: 32);f) Forward primer: CTTCTGGTTTGGTGTCAACAG (SEQ ID NO: 34); and Reverse primer: GCCTGAGATGGCAGATTTAATG (SEQ ID NO: 35);g) Forward primer: AAGAGGGCCTCCATGTTTC (SEQ ID NO: 37); and Reverse primer: CCCACGGAGTTTATCCTGAC (SEQ ID NO: 38);h) Forward primer: CCACTCTATGCGAACCACAA (SEQ ID NO: 40); and Reverse primer: CCAGGCTCAGGAGAGAGAA (SEQ ID NO: 41);i) Forward primer: CATCCACCATGGAGGTCATT (SEQ ID NO: 43); and Reverse primer: TTGAGGCACCTCGAAACAT (SEQ ID NO: 44); andj) Forward primer: AGGCGCATCCCACTCTAT (SEQ ID NO: 46); and Reverse primer: TGAGGGCACAGTCAGTAGT (SEQ ID NO: 47); and / orthe nucleotide sequence of the probe for detecting the TRPM2 gene is selected from the group consisting of:k) TGTGAAGCGGATGATGAAGGACGT (SEQ ID NO: 21);I) TGGTCCTCAGAGGAATCTCCACC (SEQ ID NO: 24);m) TGATCCAGCAGAAACTGAGCGTGT (SEQ ID NO: 27);n) AAATGTGCATGAGCCGGAGGC (SEQ ID NO: 30);o) ATGACCCGAGGAACACGGACAATG (SEQ ID NO: 33);p) CTCACAGGAGCGTGAACCATGAGG (SEQ ID NO: 36);q) AGGTGCCTCAACATGGAGCCTT (SEQ ID NO: 39);r) CTGAGGGCACAGTCAGTAGTGAGC (SEQ ID NO: 42);s) CCCTCAGGCCTATGTCTGTGAGGA (SEQ ID NO: 45); andt) TTCTGGAGGAGGGTCTTGTGGTTC (SEQ ID NO: 48).

21. The kit of any one of claims 16 to 20, which further comprises one or both of the following internal control primer pairs:Reverse primer:AGGTACTGGTCCTTATCCAGAG (SEQ ID NO: 14); and / orb) Forward primer: GTCACTTCGTGGCTAAGGTAAG (SEQ ID NO: 16); andReverse primer: CTCAGCCCTGACACGTTAATAC (SEQ ID NO: 17).

22. The kit of claim 21, which further comprises at least one probe having a sequenceselected from:AGCGTGGACTTCAGTGCATTCATGA (SEQ ID NO: 15); andACACAGACTCACCAAGCCACAGAC (SEQ ID NO: 18).

23. A method of predicting COVID-19 infectiousness and treating a subject who is infectious, the method comprising:determining expression of the TBL1XR1 gene in a biological sample from asubject having or possibly having COVID-19;optionally also detecting expression of at least one of the PPP3CB, TRPM2 and MIR4323 genes;wherein the level of expression of TBL1XR1 or at least one of the other genes indicates the likely infectiousness of the subject; andadministering a therapeutically effective amount of a COVID-19 treatment to an infectious subject.47

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