Short viral RNAS (svrnas) with theragnostic potential in infection with SARS-COV-2 and related viruses

Abstract

Provided herein are short viral RNA (svRNA) and human RNA sequences that exhibit modulated expression during viral infection and are therefore useful for detecting viral infections. Further disclosed herein are compositions, methods, and kits for detecting and treating infections, including early-stage and latent infections with low viral titers. In certain embodiments the compositions, methods, and kits for detecting and treating infections include detecting in a sample from a subject 10-50 nucleotide length short viral RNA (svRNA), modulated expression of 10-50 nucleotide length RNA in the subject, or a combination thereof, thereby detecting infection by a virus in the subject.

Description

SHORT VIRAL RNAS (SVRNAS) WITH THERAGNOSTIC POTENTIAL IN INFECTION WITH SARS-COV-2 AND RELATED VIRUSESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 613,488, filed December 21, 2023. The content of the prior application is considered part of and is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing xml file, name JHU4700- 1WO, was created on December 16, 2024, and is 85,908 bytes.BACKGROUND OF THE INVENTIONFIELD OF THE INVENTION

[0003] The present disclosure relates generally to methods of detecting viral infections and more specifically to a method of detecting modulated expression of short noncoding viral and host ribonucleic acids (RNAs) to ascertain infection status.BACKGROUND INFORMATION

[0004] The 2019 outbreak of SARS-CoV-2, the causative agent of coronavirus disease COVID-19, led to more than six million deaths by the year 2022. Amidst unprecedented mobilization of resources for diagnosis and treatment, it remains crucial to develop a more detailed understanding of the molecular perturbations accompanying SARS-CoV-2 infection. Most transcriptomic studies of infection with SARS-CoV-2 and related viruses focus on long RNA biotypes rather than small RNAs. Precise characterization of the host and viral small RNA landscape in infected cells could provide important insights. Small RNA-related molecular mechanisms that regulate the viral life cycle could be targeted for therapeutic interventions. Meanwhile, the relative stability of small RNAs could enable novel diagnostic and prognostic tools, for example, to detect the presence of SARS-CoV-2 in biological fluids and mucosal surfaces for “as-early-as-possible” diagnosis or detection of low-level virus persistence.SUMMARY OF THE INVENTION

[0005] The present disclosure is based on the seminal discovery that viral infections modulate expression of multiple short non-coding RNA (sncRNA) biotypes. While previous analyses focused on coding and regulatory transcripts (e.g., miRNA), it was surprisingly discovered that non-coding and non-regulatory sncRNAs expression is considerably more responsive to infection, and thus offers reliable biomarkers for early and latent disease detection.

[0006] Leveraging this discovery, in one embodiment, the present disclosure provides a method that includes: a) detecting in a sample from a subject: i) 10-50 nucleotide length short viral RNA (svRNA), ii) modulated expression of 10-50 nucleotide length RNA in the subject, or iii) a combination thereof, thereby detecting infection by a virus in the subject. Optionally, a therapeutic agent is administered to the subject, thereby treating the infection in the subject.

[0007] In one aspect, the virus is a positive-strand RNA virus. In some aspects, the virus is a coronavirus, a balfinivirus, a betaacoronavirus, a cadicivirus, a cardiovirus, a cosavirus, a flavivirus, a gammacoronavirus, a hepacivirus, a hepatovirus, a kobuvirus, a kobuvirus, a lagovirus, a megrivirus, a nebovirus, a norovirus, a parechovirus, a pegivirus, a rubivirus, a salivirus, a sapelovirus, a sapovirus, a senecavirus, a teschovirus, a tremovirus, a vesivirus, an aichivirus, an alphacoronavirus, an alphavirus, an aphthovirus, an avihepatovirus, an enterovirus, an erbovirus, or a torovirus, or a teschovirus.

[0008] In a further aspect, the virus is a coronavirus.

[0009] In a particular aspect, the virus is SARS-CoV-1 or SARS-CoV-2.

[0010] In certain aspects, the svRNA maps to a positive strand of a genome of the virus. In additional aspects, the svRNA maps to the human genome. In some aspects, the detecting includes measuring a prevalence of at least about 1000 counts per million, at least about 2000 counts per million, at least about 3000 counts per million, at least about 4000 counts per million, at least about 5000 counts per million, at least about 6000 counts per million, at least about 7000 counts per million, at least about 8000 counts per million, at least about 9000 counts per million, at least about 10000 counts per million, at least about 12000 counts per million, at least about 15000 counts per million, at least about 20000 counts per million, or at least about 25000 counts per million of the svRNA.

[0011] In a particular aspect, the svRNA includes one or more sequences selected from SEQ ID NOs: 12-19.

[0012] In further aspects, the RNA from the subject includes a fragment of mRNA, IncRNA, vault RNA, tRNA, or Y-RNA. In some aspects, the RNA from the subject includes one or more sequences selected from SEQ ID NOs:20-39.

[0013] In some aspects, the svRNA, the RNA from the subject, or the combination thereof includes between 2 and 10 sequences, between 2 and 20 sequences, between 2 and 30 sequences, between 2 and 40 sequences, between 2 and 50 sequences, between 2 and 75 sequences, between 2 and 100 sequences, between 2 and 200 sequences, between 2 and 500 sequences, between 5 and 10 sequences, between 5 and 20 sequences, between 5 and 40 sequences, between 5 and 50 sequences, between 5 and 75 sequences, between 5 and 100 sequence, between 5 and 200 sequences, between 5 and 500 sequences, between 10 and 20 sequences, between 10 and 30 sequences, between 10 and 40 sequences, between 10 and 50 sequences, between 10 and 75 sequences, between 10 and 100 sequences, between 10 and 200 sequences, between 10 and 500 sequences, between 20 and 30 sequences, between 20 and 40 sequences, between 20 and 50 sequences, between 20 and 100 sequences, between 20 and 200 sequences, between 20 and 500 sequences, between 30 and 50 sequences, between 30 and 100 sequences, between 30 and 200 sequences, between 30 and 500 sequences, between 50 and 100 sequences, between 50 and 200 sequences, between 50 and 500 sequences, between 100 and 200 sequences, between 100 and 500 sequences, or between 200 and 500 sequences.

[0014] In some aspects, the svRNA, the RNA from the subject, or the combination thereof has a length of about 15-26 nucleotides. In further aspects, the svRNA, the RNA from the subject, or the combination thereof includes multiple RNA biotypes. In specific aspects, an RNA molecule of the svRNA, the RNA from the subject, or the combination thereof does not comprise a 5’-phosphate, a 3’-hydroxyl, or a combination thereof.

[0015] In another aspect, the sample includes a cell lysate or a bronchial fluid. In an additional aspect, the detecting includes reverse transcription, polymerase chain reaction (PCR), real-time PCR (rt-PCR), quantitative PCR (q-PCR), nucleic acid sequence-base amplification (NASB A), ligase chain reaction, multiplex ligatable probe amplification, rolling circle amplification, in- vitro transcription (IVT), strand displacement amplification, fluorescence in situ hybridization (FISH), transcription-mediated amplification (TMA), Eberwine amplification, microarray detection, electrophoresis, high pressure liquid chromatography (HPLC), affinity chromatography, CRISPR-based detection, or a combination thereof. In a further aspect, the detecting includes ligating an adapter, a barcode, or a polyadenylation sequence to the svRNA, the RNA from the subject, or the combination thereof. In a specific aspect, the detectingincludes use of a primer that includes a sequence selected from SEQ ID NOs: l-l l. In a particular aspect, the detecting includes reverse transcription of the svRNA, the RNA from the subject, or the combination thereof to generate DNA, and detecting a sequence of the DNA. In a further aspect, the detecting includes separating the svRNA, the RNA from the subject, or the combination thereof from the sample from the subject.

[0016] In an additional aspect, the detecting includes separating the svRNA, the RNA from the subject, or the combination thereof from nucleic acids with lengths greater than about 50 nucleotides, greater than about 75 nucleotides, greater than about 100 nucleotides, greater than about 150 nucleotides, greater than about 200 nucleotides, or greater than about 250 nucleotides. In one such aspect, the separating includes filtration, extraction, precipitation, or a combination thereof. In a particular aspect, the detecting includes: i) polyadenylating the svRNA, the RNA from the subject, or the combination thereof, thereby generating polyadenylated RNA, ii) reverse transcription of the polyadenylated RNA, thereby generating DNA, and iii) quantitative PCR of the DNA.

[0017] In some aspects, the method includes administering a therapeutic agent selected from an antiviral agent, a vaccine, a vaccine adjuvant, an immune stimulant, an antibody, a toll-like receptor agonist, a cytokine, a chemokine, an interleukin, an oligodeoxynucleotide, a glucan, a type I interferon receptor agonist, a type II interferon receptor agonist, an anti-inflammatory agent, a nuclease, a cell-based therapy, or a combination thereof. In particular aspects, the antiviral agent includes ABT-263, alisporivir, amiodarone, arbidol, bevacizumab, boceprevir, calpain inhibitor II, calpain inhibitor XII, camostat, camphor, carvedilol, cepharanthine, chloroquine, colchicine, corticosteroids, cyclosporine, dactinomycin, dalbavancin, dasatinib, dexamethasone, eflornithine, emetine, emodin, emodin, eplerenone, equilin, famotinine, favilavir, favipiravir, flavipavir, flovoxamine, galdecivir, GC-376, gemcitabine, glycyrrhizin, GS-441524, hexachlorophene, homoharringtonine, hydroxychloroquine, indomethacin, irbesartan, ivermectin, lopinavir, lopinavir, luteolin, mefloquine, memantine, mercaptopurine, mesalazine, monesin, nafamosta, nelfinavir, niclosamide, nitazoxanide, oritavancin, oxymetholone, paroxetine, promethazine, quinacrine, rapamycin, ribavirin, ritonavir, ritonavir, saracatinib, sarilumab, silvestrol, sirolimus, solnatide, teicoplanin, telavancin, tilorone, tocilizumab, toremifene, trametinib, umifenovir, or remdesivir.

[0018] In some aspects, the subject is asymptomatic of the infection. In other aspects, the subject received a chest X-ray (SCR) score of less than about 25, of less than about 20, of less than about 15, of less than about 12, of less than about 10, of less than about 8, of less thanabout 6, of less than about 5, of less than about 4, or of less than about 3. In another aspect, the infection is a chronic infection.

[0019] In some aspects, the modulated expression is expression relative to a control sample from a non-infected subj ect.

[0020] In another embodiment, the present disclosure provides a method of identifying a subject as a likely responder to antiviral therapy that includes: a) detecting in a sample from the subject: i) short viral RNA (svRNA) with a length of about 10-50 nucleotides, ii) modulated expression of 10-50 nucleotide RNA in the subject relative to a control sample, or iii) a combination thereof; and b) classifying the subject: as a likely responder to antiviral therapy based on the svRNA detected in the sample, the modulated expression of the RNA in the subject relative to the control sample, or the combination thereof, thereby identifying the subject as a likely responder to antiviral therapy.

[0021] In one aspect, the detecting includes use of a primer that includes a sequence selected from SEQ ID NOs: 1-11. In another aspect, the svRNA includes a sequence selected from SEQ ID NOs: 12-19. In a further aspect, the RNA in the subject includes a sequence selected from SEQ ID NOs:20-39.

[0022] In some aspects, the control sample is a sample from a non-responder to the antiviral therapy, a sample from a subject that does not have a viral infection, or a combination thereof. In further aspects, the classifying includes determining that the subject has a viral infection.

[0023] In a further aspect, the viral infection is infection with a positive-strand RNA virus. In a particular aspect, the viral infection is a coronavirus infection. In a specific aspect, the viral infection is a SARS-CoV-1 or SARS-CoV-2 infection.

[0024] In another aspect, the classifying includes determining the severity of the viral infection. In a particular aspect, the detecting includes quantifying the svRNA, quantifying a degree of the modulated expression of the RNA in the subject, or the combination thereof in the sample from the subject.

[0025] In an additional aspect, the present disclosure provides a kit for performing a method of the present disclosure.

[0026] In a further embodiment, the present disclosure provides a composition with a nucleic acid that includes a sequence selected from SEQ ID NOs: 1-11. In some aspects, the composition includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or 11 sequences selected from SEQ ID NOs: 1-11.

[0027] One aspect provided herein includes a kit that contains a composition of the present disclosure. In some aspects, the kit includes: i) a lysis buffer, ii) a DNAse, iii) a reverse transcriptase, iv) a universal primer, v) a dNTP, vi) a filter, vii) a ligase, viii) an adapter, barcode, or polyadenylation sequence, ix) a multiwell plate, or x) a combination thereof. In particular aspects, two or more wells of the multiwell plate comprise different primers selected from SEQ ID NOs: l-l l.

[0028] Further aspects provided herein include a method of using a kit of the present disclosure that includes detecting RNA in a biological sample.

[0029] In another embodiment, the present disclosure provides a method of detecting infection by a virus in a subject that includes: a) detecting 10-50 nucleotide length short viral RNA (svRNA), modulated expression of 10-50 nucleotide length RNA in the subject, or a combination thereof, by: i) polyadenylating the svRNA, the RNA from the subject, or the combination thereof, thereby generating polyadenylated RNA, ii) reverse transcribing the polyadenylated RNA, thereby generating DNA, and iii) performing quantitative PCR on the DNA, thereby detecting the svRNA, the modulated expression of the RNA in the subject, or the combination thereof; thereby detecting the infection by the virus in the subject.

[0030] In an additional embodiment, the present disclosure provides a method that includes: a) detecting in a sample from a subject: i) RNA that includes a sequence selected from SEQ ID NOs: 12-19 and a length of 10-50 nucleotides, ii) modulated expression of RNA comprising a sequence selected from SEQ ID NOs:20-39 and a length of 10-50 nucleotides, or iii) a combination thereof, thereby detecting infection by a virus in the subject.

[0031] In some aspects, the method further includes administering a therapeutic agent to the subject, thereby treating the infection in the subject.

[0032] In one aspect, the virus is SARS-CoV-1 or SARS-CoV-2. In an additional aspect, the detecting includes the use of a primer that includes a sequence selected from SEQ ID NOs: l- 11.

[0033] In a further embodiment, the present disclosure provides a method of determining the severity of a viral infection in a subject that includes: a) quantifying in a sample from the subject: i) an amount of short viral RNA (svRNA) with a length of about 10-50 nucleotides, ii) a degree of modulated expression of 10-50 nucleotide RNA in the subject relative to a control sample, or iii) a combination thereof; and b) determining the severity of the viral infection based on the svRNA detected in the sample, the modulated expression of the RNA in thesubject relative to the control sample, or the combination thereof, thereby determining the severity of the viral infection in the subject.

[0034] In some aspects, the quantifying includes determining a change in the amount of the svRNA in the sample, a change in a degree of the modulated expression of the 10-50 nucleotide RNA in the subject relative to the control sample, or a combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1A-1C illustrate a set of tables and figures that show overexpression of SARS- CoV-1 and SARS-CoV-2 derived short viral RNAs in infected Calu3 cells. FIG. 1A is a table and schematic that shows accession numbers of small RNA-seq datasets and the parameters used for reads alignment to corresponding viral genome references. FIG. IB is a set of graphs showing expression of viral small RNA reads (in count-per-million metrics) in Calu3 cells infected with either SARS-CoV-1 or SARS-CoV-2 at 4 hours (14), 12 hours (112) and 24 hours (124) post-infection. The viral RNA content in mock (uninfected) cells at 4 hours (M4) and 24 hours (M24) are also shown. FIG. 1C is a table that shows percentage of total small RNA-seq reads aligned to forward and reverse strands of SARS-CoV-1 and SARS-CoV-2 genomes calculated at 24 hours post-infection. Note: a certain percentage of reads from SARS-CoV-1 infected cells aligned to the SARS-CoV-2 genome and vice versa.

[0036] FIG. 2A-2C illustrate a set of plots and a table of short RNAs derived from SARS- CoV-2. FIG. 2A illustrates a set of genomic IGV map of reads aligned to the SARS-CoV-2 genome generated from small RNA-seq datasets of Calu3 cells 24 hours post infection. The five most overrepresented short viral RNAs are highlighted by vertical rectangles. FIG. 2B illustrates a genomic IVG map showing the regions with highest coverage towards two motif sequences from total reads and SARS-CoV-2 reads only. FIG. 2C illustrates a table with a list of highly overrepresented viral sequences selected for RT-qPCR validation and their copy numbers in small RNA-seq datasets from Calu3 cells 24 hours post-infection with SARS-CoV- 2.

[0037] FIGS. 3A-3C illustrate a set of schematics and plots showing experimental validation of several identified svRNAs in infected A549 cells. FIG. 3A is a schematic of an experimental workflow showing SARS-CoV-2 infection and RNA isolation. FIG. 3B is a schematic showing a RT-qPCR workflow used for short RNAs detection. FIG. 3C is a series of graphs showing Cq values for the analyzed short viral RNAs in uninfected vs. infected A549 cells.

[0038] FIGS. 4A-4D illustrate a workflow and set of plots, diagrams, and tables of small RNA-seq data analysis of Calu-3 cells 24-hour post-infection with SARS-CoV-1 and SARS- CoV-2. FIG. 4A illustrates a sequential reads mapping workflow used to generate count tables from raw small RNA-seq data; FIG. 4B illustrates (top plot) a multidimensional scaling (MDS) plot of log-CPM values over dimensions 1 and 2 with samples shaded and labeled by sample showing distances between the compared groups of samples (upper) and (bottom diagram) a Venn diagram showing the number of DE genes in the comparison between SARS-CoV-1 infected cells vs. mock control (left), SARS-CoV-2 infected cells vs. mock control (right), and the number of genes that are DE in both comparisons (center). The number of genes that are not DE in either comparison are marked in the bottom-right; FIG. 4C illustrates a set of meandifference (MD) plots of differentially expressed genes at small RNA level between mock and SARS-CoV-1 (upper plot)) as well as mock vs. SARS-CoV-2 (lower plot) infected Calu-3 cells FIG. 4D is a set of tables summarizing the number of differentially expressed transcripts for each RNA biotype.

[0039] FIGS. 5A-5D illustrate a table, schematic, and set of plots showing alignment of short viral RNA reads to human sequences. FIG. 5A is a schematic of an RNA-seq read mapping workflow and parameters. FIG. 5B illustrates a set of tables showing the percentage of total viral small RNA reads (from both forward and reverse strands) aligned to certain hg38 sequences in forward and reverse orientations. FIG. 5C illustrates a plot of combined length distributions of SARS-CoV-1 and SARS-CoV-2 reads mapped to human mRNA and IncRNA references. FIG. 5D is a plot of combined length distributions of SARS-CoV-1 and SARS- CoV-2 reads mapped to human introns and intergenic regions.

[0040] FIG. 6 illustrates a set of IGV maps showing reads coverage towards SARS-CoV-1 and SARS-CoV-2 genomes in small RNA-seq datasets from Calu-3 cells 24-hours post infection with each virus. Reads mapped to forward (top plots) and reverse (bottom plots) strands are shown separately.

[0041] FIG. 7 illustrates a set of IGV coverage maps of reads aligned towards SARS-CoV-2 genome only in small RNA-seq datasets from Calu-3 cells 4-hours, 12-hours and 24-hours post infection with SARS-CoV-2. Reads mapped to forward and reverse strands are shown separately in the top and bottom set of plots, respectively.

[0042] FIGS. 8A-8C illustrate a set of plots of length distributions of reads mapped to multiple RNA biotypes from Calu-3 cells 24-hour post-infection with SARS-CoV-2. FIG. 8A illustrates a plot of length distributions of RNY. FIG. 8B illustrates a plot of lengthdistributions of vault RNA. FIG. 8C illustrates a plot of length distributions of tRNA. The top 5 overrepresented reads are shown for each RNA biotype.

[0043] FIG. 9 illustrates a set of plots of IGV coverage maps of reads aligned to the main human RNY transcripts in small RNA-seq datasets from Calu3 cells mock infected and at 24- hours post-infection with SARS-CoV-2.

[0044] FIG. 10 illustrates a set of plots of IGV coverage maps of reads aligned to human vault RNA transcripts in small RNA-seq datasets from Calu-3 cells 24-hours post infection with SARS-CoV-2.

[0045] FIG. 11 illustrates a set of plots of IGV coverage maps of reads aligned to the top four upregulated tRNA transcripts in small RNA-seq datasets from Calu3 cells mock infected and at 24-hours post-infection with SARS-CoV-2.

[0046] FIG. 12 illustrates a plot of length distributions of reads mapped to miscellaneous RNY references from GENCODE v.41 and the top 5 overrepresented reads.

[0047] FIGS. 13A-13F illustrate graphs that show expression of three svRNAs in bronchial fluid collected from individuals diagnosed with SARS-CoV-2 during acute phase. FIG. 13A is a graph showing normalized qPCR results (fold-change, FC) for 29096F svRNA. FIG. 13B is a graph showing normalized qPCR results (CT) for 29096F._FIG. 13C is a graph showing normalized qPCR results (fold-change, FC) for 245 IF svRNA. FIG. 13D is a graph showing normalized qPCR results (CT) for 245 IF. FIG. 13E is a graph showing normalized qPCR results (fold-change, FC) for 3178F svRNA. FIG. 13F is a graph showing normalized qPCR results (CT) for 3178F.DETAILED DESCRIPTION OF THE INVENTION

[0048] The present disclosure is based on the seminal discovery that viral infections modulate expression of multiple short non-coding RNA (sncRNA) biotypes.

[0049] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0050] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, forexample, references to “the method” includes one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0051] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0052] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0054] The present disclosure provides viral RNAs and modulations in human short RNA expression useful for detecting viral infection. While it was shown herein that SARS-CoV-1 and SARS-CoV-2 microRNA levels increase minimally upon infection, it was surprisingly found that levels of other RNA biotypes exhibit pronounced variations in expression levels during infection. In particular, the foregoing analyses detail quantifiable responses of small RNA expression during SARS-CoV-1 and SARS-CoV-2 infection. Furthermore, these analyses show that SARS-CoV-2 induces differential expression of a dramatically larger number of small RNAs as compared with SARS-CoV-1. Importantly, many of these small RNA fragments were align to protein-coding and IncRNA genes. At the same time, despite robust changes in small RNA expression, only a few miRNAs were affected, suggesting a limited role of host miRNAs in the progression of infection.

[0055] Leveraging these discoveries, the present disclosure provides compositions, kits, and methods for detecting viral infections by measuring virus-derived svRNAs and / or modulation of host RNA expression as sensitive biomarkers for early diagnosis or detection of infection. In one embodiment, the present disclosure provides a method that includes: a) detecting in a sample from a subject: i) 10-50 nucleotide length short viral RNA (svRNA), ii) modulated expression of 10-50 nucleotide length RNA in the subject, or iii) a combination thereof, thereby detecting infection by a virus in the subject. Optionally, a therapeutic agent is administered to the subject, thereby treating the infection in the subject.

[0056] The terms “administration of’ and or “administering” should be understood to mean providing a compound or pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. As non-limiting examples, administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, nebulization, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extraamniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intrabursal, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebroventricular, intracistemal, intracorneal, intracoronal, intracoronary, intracorpous cavemaosum, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intrapleural, intraprostatic, intrapulmonary, intrasinal, intrasynovial, intratendinous, intratesticular, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasogastric, ophthalmic, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, retrobulbar, subconjunctival, sublingual, submucosal, topical, transmucosal, transplacental, transtympanic, ureteral, urethral, infraorbital, intraparenchymal, intraventricular, stereotactic administration subcuticular, or any combination thereof. A form of administration can be tailored for a particular infection based on the properties and localization of the infection and drug.

[0057] The terms “therapeutically effective amount”, “effective dose,” “therapeutically effective dose”, “effective amount,” or the like refer to that amount of the subject agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome (e.g., treatment of the disease). Such amount should be sufficient to eliminate infected cells. The effective amount can be determined as described herein.

[0058] As used herein the term “biomarker” refers to a peptide, a protein, a nucleic acid, an antibody, a gene, a metabolite, or any other substance used as an indicator of a biologic state. It is a characteristic that is measured objectively and evaluated as a cellular or molecular indicator of pathological processes, or of normal biological processes, or pharmacologic response to a therapeutic intervention. In some embodiments, a biomarker refers to a nucleic acid. In some embodiments, a biomarker refers to a ribonucleic acid (RNA). In some embodiments, a biomarker refers to a short viral RNA (svRNA) that is 15-26 ribonucleotides in length. In some embodiments, a biomarker refers to a ribonucleic acid that is 10-50 ribonucleotides in length.

[0059] As used herein, the terms “detecting”, “determining”, and their other grammatical forms, are used to refer to methods performed for the identification or quantification of a biomarker; for example, they can refer to methods performed for the identification or quantification of the presence or level of svRNA in a biological sample from a subject. The amount of biomarker expression or activity detected in the sample can be none, or it can be below the level of detection of the assay or method used.

[0060] The term “infection” as used herein refers to the initial entry of a pathogen into a host; and the condition in which a pathogen has become established in or on cells or tissues of a host; such a condition does not necessarily constitute or lead to a disease. Examples of pathogens may include but are not limited to a virus, bacteria, and fungus.

[0061] As used herein the term “infection by a virus” refers to when a virus enters a host organism, and begins to replicate within the host’s cells, causing disruption to normal bodily functions and potentially leading to illness with symptoms like fever, cough, or fatigue.

[0062] The terms “infectious agent” or “causal agent” are used interchangeably to refer to a pathogen that causes an infection.

[0063] The term “inhalation” as used herein refers to the act of drawing in a medicated vapor with the breath.

[0064] The term “inhalation delivery device” as used herein refers to a machine / apparatus or component that produces small droplets or an aerosol from a liquid or dry powder aerosol formulation and is used for administration through the mouth in order to achieve pulmonary administration of a drug, e.g., in solution, powder, and the like. Examples of inhalation delivery device include, but are not limited to, a nebulizer, a metered-dose inhaler, and a dry powder inhaler (DPI).

[0065] The term “infuse” and its other grammatical forms as used herein refers to introduction of a fluid other than blood into a vein.

[0066] The term “microRNA,” “miRNA”, or “miR” as used herein refers to a class of small, non-coding RNA molecules, usually from about 18 to about 28 nucleotides in length. MicroRNAs are partially complementary to one or more messenger RNA (mRNA) molecules, and function in posttranscriptional regulation of gene expression and RNA silencing. MicroRNAs typically contain 5 ’-phosphate and 3 ’-OH groups, are associated with proteins of the Argonaute family, and have a well-characterized mechanism of generation. Mature microRNAs are often derived from stem-loop containing regions of precursor nucleic acids, thereby differing from many of the svRNAs disclosed herein, which lack such structures.

[0067] The terms “polynucleotide”, “polynucleotide sequence”, and “nucleic acid sequence” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of DNA or RNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (ribonucleotides or deoxyribonucleotides) can be referred to by a single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate (for RNA or DNA, respectively), “G” for guanylate or deoxyguanylate (for RNA or DNA, respectively), “U” for uridylate (for RNA), “T” for deoxythymidylate (for DNA), “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, “W” for A or T, and “N” for any nucleotide (e.g., N can be A, C, T, or G, if referring to a DNA sequence; N can be A, C, U, or G, if referring to an RNA sequence). Any RNA sequence (e.g., svRNA) disclosed herein may be encoded by a suitable DNA sequence.

[0068] As used herein, the term “subject” refers to any individual or patient to which the disclosed methods are performed, to whom the disclosed compositions are administered, or from whom a biological material (e.g., the sample from the subject in the disclosed methods) is obtained. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be a non-human animal. Thus, other animals, including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0069] The term "treatment" is used interchangeably herein with the term "therapeutic method" or “therapy” and refers to 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic conditions or disorder (e.g., idiopathic pulmonary fibrosis), and / or 2) prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventive measures).

[0070] As used herein, the terms “wild type,” “naturally occurring,” or grammatical equivalents thereof, are meant to refer to an amino acid sequence or a nucleotide sequence that is found in nature and includes allelic variations; that is, an amino acid sequence or a nucleotide sequence that usually has not been intentionally modified. Accordingly, the term “non- naturally occurring,” “synthetic,” “recombinant,” or grammatical equivalents thereof, are used interchangeably to refer to an amino acid sequence or a nucleotide sequence that is not found in nature; that is, an amino acid sequence or a nucleotide sequence that usually has been intentionally modified. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations.

[0071] As used herein, the term “modulated expression” is meant to refer to detectable changes in expression. A detectable change in expression can be relative to a control sample, such as an identical type of sample from another subject or a sample previously collected from the subject of a method disclosed herein. A detectable change in expression can also be relative to a database or standard value. As non-limiting examples, a threshold for modulated expression can be a 1.25-fold change, a 1.5-fold change, a 2-fold change, a 2.5-fold change, a 3-fold change, a 4-fold change, a 5-fold change, or a 10-fold change in a level of expression. In many aspects, a threshold for modulated expression is a 2-fold change in a level of expression.

[0072] As used herein the term “therapeutic agent” or “drugs” refers to substances that are administered to influence the outcome of the disease, whether to cure it, to reduce, or to eliminate some of its symptoms, or simply to improve the quality of life for the patient in question.

[0073] In many aspects, the svRNA, the RNA from the subject, or the combination thereof includes small noncoding RNA (sncRNA). sncRNAs are known to mediate diverse regulatory functions during cell proliferation, differentiation, apoptosis and response to infection. MicroRNAs, piwi-interacting RNAs and tRNA-derived RNA fragments are some examples ofthe most widely studied sncRNAs. However, it was surprisingly shown herein that the svRNAs that are highly expressed during infection are much more abundant than viral miRNAs such as CvmiR-5, CoV2-miR-O7a.1, and CoV2-miR-O7a.2. In particular, these viral miRNAs were 100-1000-fold lower than svRNAs derived from the sense viral RNA strand and 10-100-fold lower than svRNAs derived from the antisense strand, suggesting higher diagnostic potential for the svRNAs disclosed herein. Without being bound by theory, it is hypothesized herein that much more subtle expression of virus-encoded miRNAs could result from slower maturation of Argonaute-associated RNAs as compared with other svRNAs and / or higher stability of the latter.

[0074] Cells infected with different viruses including SARS-CoV-1 and SARS-CoV-2 exhibit changes in host microRNA expression accompanied by subsequent deregulation of microRNA target genes. However, differential expression of other small RNA species was previously poorly addressed, presumably due to the fact that widely used protocols for small RNA sequencing library preparation preferentially incorporate RNAs containing 5 ’-Phosphate and 3 ’-OH groups.

[0075] As used herein, the terms “short viral RNA” and “svRNA” denotes short viral genomic RNA or virus-derived transcripts. The data disclosed herein suggest that viruses encode their own sncRNAs to regulate replication, transcription, and translation of viral genomes as well as to influence immune evasion and inflammatory response. As a non-limiting example, svRNA can have a length of less than or equal to about 50 nucleotides, less than or equal to about 40 nucleotides, less than or equal to about 30 nucleotides, or less than or equal to about 26 nucleotides. In many cases, svRNA of the present methods is not miRNA.

[0076] As used herein the term “small noncoding RNAs” or “sncRNAs” refers to a type of RNA molecule that is less than 200 nucleotides in length and plays a vital role in cellular processes. Non limiting examples of sncRNAs include microRNA (miRNA), small interfering RNAs (siRNAs), pi wi -interacting RNAs (piRNAs), small nucleolar RNA (snoRNA), transfer RNA (tRNA), tRNA-derived small RNAs, ribosomal RNA fragments (rRFs), small nuclear RNA (snRNA), Y-RNA, and vault RNA (vtRNA).

[0077] In one aspect, the virus is a positive-strand RNA virus. For example, the virus can be a balfinivirus, a betaacoronavirus, a cadicivirus, a cardiovirus, a coronavirus, a cosavirus, a flavivirus, a gammacoronavirus, a hepacivirus, a hepatovirus, a kobuvirus, a kobuvirus, a lagovirus, a megrivirus, a nebovirus, a norovirus, a parechovirus, a pegivirus, a rubivirus, a salivirus, a sapelovirus, a sapovirus, a senecavirus, a teschovirus, a tremovirus, a vesivirus, anaichivirus, an alphacoronavirus, an alphavirus, an aphthovirus, an avihepatovirus, an enterovirus, an erbovirus, or a torovirus, or a teschovirus. In particular aspects of the present disclosure, the virus is a coronavirus.

[0078] In some aspects, the virus is SARS-CoV-1 or SARS-CoV-2. SARS-CoV-1 and SARS-CoV-2 are viruses with positive-sense single-stranded RNA genomes that utilize translational machinery of the host cell to produce proteins necessary for replication of viral RNA and assembly of viral particles. Host cells detect and counter these pathogens through pathways of the innate antiviral immune response such as dsRNA-mediated activation of Tolllike receptors. SARS-CoV-1 and SARS-CoV-2 replication both involve synthesis of various RNA intermediates whose stability and diagnostic potential remains largely unexplored. Current PCR-based methods for COVID-19 diagnostics rely on certain amplicons within the full-length viral RNA.

[0079] In certain aspects, the svRNA maps to a positive strand of a genome of the virus. While many positive- strand RNA viruses generate complementary negative-strand copies of their genomes during replication, a surprising discovery herein is that positive- strand RNA viral infection generates an excess of positive strand (relative to negative strand) svRNAs, allowing positive strand svRNA to serve as a sensitive marker for early viral detection. For example, FIG. IB shows that SARS-CoV-1 and SARS-CoV-2 positive strand svRNA levels are readily distinguishable from negative svRNA levels 12 hours after infection. Without being bound by theory, it is postulated herein that the excess of positive strand svRNA during infection may be due to greater protection of (+) genome strands by ribosomes or other associating proteins.

[0080] In other aspects, the svRNA maps to the human genome. It was shown herein that a significant proportion of short viral RNA reads from both SARS-CoV-1 and SARS-CoV-2 infected cells also aligned to human transcriptome and genome references with perfect complementarity. Such cross-mapping could result from integration events during evolution, in a similar to those that occurred for human endogenous retroviruses. The presence of the relatively high proportion of short RNAs derived from viral genome that are similar to human sequences might indicate the existence of yet uncharacterized mechanism of gene expression regulation in the host cells mediated by SARS-CoV-1 and SARS-CoV-2.

[0081] In some aspects, the detecting includes measuring a prevalence of at least about 1000 counts per million, at least about 2000 counts per million, at least about 3000 counts per million, at least about 4000 counts per million, at least about 5000 counts per million, at leastabout 6000 counts per million, at least about 7000 counts per million, at least about 8000 counts per million, at least about 9000 counts per million, at least about 10000 counts per million, at least about 12000 counts per million, at least about 15000 counts per million, at least about 20000 counts per million, or at least about 25000 counts per million of the svRNA.

[0082] In a particular aspect, the svRNA includes one or more sequences selected from SEQ ID NOs: 12-19.

[0083] In some aspects, the detecting comprises converting the svRNA, the RNA from the subj ect, or the combination thereof into DNA (e.g., through reverse transcription) and detecting the DNA. Accordingly, in some aspects, the detecting comprises detecting a corresponding DNA sequence of the svRNA, the RNA from the subject, or the combination thereof, such as optionally barcoded cDNA generated from the svRNA, the RNA from the subject, or the combination thereof. For example, detecting the svRNA can include detecting one or more of SEQ ID NOs:40-47, and detecting modulated expression of the RNA from the subject can include detecting one or more of SEQ ID NOs:48-67.

[0084] It was further determined herein that virus infected cells exhibited modulated expression for numerous RNA biotypes. In particular, only a small number of host miRNAs were upregulated in infected cells. While several dozen miRNAs and premiRNAs were statistically significantly downregulated after SARS-CoV-2 infection, only a few showed 4- fold or greater changes. Such a limited response of the miRNA transcriptome was unexpected given the dramatic impact of both SARS-CoV-1 and SARS-CoV-2 on viability and the mRNA transcriptome of Calu-3 cells. Up- and down-regulation of multiple small RNAs aligned to human protein-coding mRNA and IncRNA transcripts in the infected Calu-3 cells versus mock controls. It is feasible that these RNAs could possess certain regulatory roles but might also persist as simple by-products of long RNA degradation and the background transcription level.

[0085] However, changes in host microRNA expression in SARS-CoV-1 and SARS-CoV-2 infected cells were accompanied by subsequent deregulation of microRNA target genes. In particular, short non-coding RNAs including Y-RNA, tRNA and vault RNA exhibited upregulation 24 hours after SARS-CoV-2 infection. Following from this discovery, a method disclosed herein can include detecting modulated expression of host mRNA, IncRNA, vault RNA, tRNA, or Y-RNA in a sample from the subject. In particular aspects, the RNA from the subject includes one or more sequences selected from SEQ ID NOs:20-39, for example at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen,at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or all twenty of SEQ ID NOs:20-39.

[0086] In particular aspects, a method disclosed herein includes detecting modulated expression of host Y-RNA. In a specific aspect, the method includes detecting RNY1, RNY3, RNY4 and RNY5 RNA. Surprisingly, SCARS-CoV-2 infected cells exhibited significant (up to 8-10-fold) upregulation of short RNAs deriving from all four human Y-RNA genes (RNY1, RNY3, RNY4 and RNY5). In addition to bona fide Y-RNAs, multiple Y-RNA pseudogenes scattered across the human genome were also differentially regulated. While the number of reads deriving from Y-RNA pseudogenes constituted only a minor fraction, certain miscellaneous transcripts demonstrated more profound upregulation as compared with the four main Y-RNA transcripts. It should be noted however, that all observed RNY reads were significantly shorter than full-length Y-RNAs and mapped predominantly to 3 ’-and 5’ - termini of the parental transcripts. Full-length Y-RNAs might not be efficiently captured by the standard reverse transcription reaction and thus remain poorly detectable by NGS. On the other hand, short Y-RNA-derived fragments are permissive for sequencing.

[0087] As used herein the term “Y-RNA” refers to a class of small, non-coding RNA molecules that are primarily known for their interaction with the protein Ro60. Y-RNAs are transcribed by RNA polymerase III and play a role in regulating DNA replication within mammalian cells, with their structure characterized by a distinct stem-loop formation. Non limiting examples of Y-RNAs include RNY1, RNY3, RNY4, and RNY5 RNA.

[0088] In further aspects, a method disclosed herein includes detecting modulated expression of RNY4 RNA. Y-RNA, particularly RNY4, is upregulated in interferon alpha (IFN-a)- stimulated B cells. IFN-a is a cytokine associated with viral infection, including by SARS- CoV-2; therefore, increased Y-RNA levels may be triggered by IFN-a that is released during infection. Interestingly, Y-RNA is triphosphorylated and can thereby trigger cellular RNA sensors such as RIG-I, leading to IFN-a production. For SARS-CoV-2, it is hypothesized herein that Y-RNA may play a role in a feed-forward loop driving antiviral cytokine expression during infection.

[0089] The svRNA, the RNA from the subject, or the combination thereof can include a single sequence. Alternatively, the svRNA, the RNA from the subject, or the combination thereof can include a plurality of sequences. For example, in some aspects, the svRNA, the RNA from the subject, or the combination thereof includes between 2 and 10 sequences, between 2 and 20 sequences, between 2 and 30 sequences, between 2 and 40 sequences,between 2 and 50 sequences, between 2 and 75 sequences, between 2 and 100 sequences, between 2 and 200 sequences, between 2 and 500 sequences, between 5 and 10 sequences, between 5 and 20 sequences, between 5 and 40 sequences, between 5 and 50 sequences, between 5 and 75 sequences, between 5 and 100 sequence, between 5 and 200 sequences, between 5 and 500 sequences, between 10 and 20 sequences, between 10 and 30 sequences, between 10 and 40 sequences, between 10 and 50 sequences, between 10 and 75 sequences, between 10 and 100 sequences, between 10 and 200 sequences, between 10 and 500 sequences, between 20 and 30 sequences, between 20 and 40 sequences, between 20 and 50 sequences, between 20 and 100 sequences, between 20 and 200 sequences, between 20 and 500 sequences, between 30 and 50 sequences, between 30 and 100 sequences, between 30 and 200 sequences, between 30 and 500 sequences, between 50 and 100 sequences, between 50 and 200 sequences, between 50 and 500 sequences, between 100 and 200 sequences, between 100 and 500 sequences, or between 200 and 500 sequences.

[0090] In some aspects, the svRNA, the RNA from the subject, or the combination thereof has a length of about 10 to 15 nucleotides, about 10 to 20 nucleotides, about 10 to 26 nucleotides, about 10 to 30 nucleotides, about 10 to 40 nucleotides, about 10 to 50 nucleotides, about 15 to 20 nucleotides, about 15 to 26 nucleotides, about 15 to 30 nucleotides, about 15 to 40 nucleotides, about 15 to 50 nucleotides, about 20 to 26 nucleotides, about 20 to 30 nucleotides, about 20 to 40 nucleotides, about 20 to 50 nucleotides, about 26 to 40 nucleotides, about 26 to 50 nucleotides. In particular aspects, the svRNA, the RNA from the subject, or the combination thereof has a length of about 15-26 nucleotides. In further aspects, the svRNA, the RNA from the subject, or the combination thereof includes multiple RNA biotypes. In specific aspects, an RNA molecule of the svRNA, the RNA from the subject, or the combination thereof does not comprise a 5’-phosphate, a 3’-hydroxyl, or a combination thereof.

[0091] For the methods disclosed herein, the sample from the subject can be any biological sample derived from the subject that contains RNA. The sample can include whole cells, cell lysate, a body fluid, whole blood, plasma, serum, bronchoalveolar lavage (BAL) fluid, bronchial fluid, cerebral spinal fluid (CSF), urine, sweat, saliva, tears, pulmonary secretions, mucous, seminal fluid, stool, cervical scraping, cysts, amniotic fluid, intraocular fluid, a nasopharyngeal swab, a buccal or nasal swab, or any combination thereof. In a particular aspect, the sample from the subject can be any biological sample derived from a deceased subj ect that contains RNA. In a particular aspect, the sample includes a cell lysate or a bronchial fluid. “Bronchial fluid” samples can be collected after a coughing procedure.

[0092] In some aspects, RNA is isolated from cell lysate or bronchial fluid. Methods for isolating RNA from cell lysate or bronchial fluid are known in the art. Examples of RNA isolation methods from cell lysate include but are not limited to organic extraction, silica column-based purification (e.g. using kits such as RNeasy), magnetic bead-based purification, and direct lysis methods. Examples of methods for isolating RNA from bronchial fluid include but are not limited to organic extraction, silica column-based purification (e.g. using kits such as RNeasy), magnetic bead-based purification, direct lysis methods, and kits used to specifically isolate and purify cell-free RNA (cfRNA) from liquid samples (e.g., blood plasma or serum) or to assess the efficiency of cell-free RNA (cf-RNA) isolation between samples.

[0093] In an additional aspect, the detecting includes reverse transcription, polymerase chain reaction (PCR), real-time PCR (rt-PCR), quantitative PCR (q-PCR), nucleic acid sequencebase amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, rolling circle amplification, in-vitro transcription (IVT), strand displacement amplification, fluorescence in situ hybridization (FISH), transcription-mediated amplification (TMA), Eberwine amplification, microarray detection, electrophoresis, high pressure liquid chromatography (HPLC), affinity chromatography, CRISPR-based detection, or a combination thereof. The detecting can also include ligating an adapter, a barcode, or a polyadenylation sequence to the svRNA, the RNA from the subject, or the combination thereof. In many aspects disclosed herein, the methods include reverse transcription to generate DNA. For example, the detecting can include polyadenylating the svRNA, the RNA from the subject, or the combination thereof, thereby generating polyadenylated RNA. In another example the detecting can include reverse transcription of the polyadenylated RNA, thereby generating DNA, and quantitative PCR of the DNA. In a specific aspect, the detecting includes use of a primer that includes a sequence selected from SEQ ID NO: 1-11 (e.g., use of a primer to amplify DNA generated from RNA from the subject). In particular aspects, the detecting includes Capture and Amplification by Tailing and Switching (e.g., as outlined in Turchinovich et al. RNA Biology, 2014, 77(7):817-828) or switching mechanism at the 5' end of the RNA transcript (e.g., as outlined in Picelli et al. Nat. Methods, 2013; 70: 1096-1098).

[0094] In a further aspect, the detecting includes separating the svRNA, the RNA from the subject, or the combination thereof from the sample from the subject, for example with extraction, precipitation, and / or filtration. In particular, the detecting can include separating the svRNA, the RNA from the subject, or the combination thereof from nucleic acids with lengths greater than about 50 nucleotides, greater than about 75 nucleotides, greater than about 100nucleotides, greater than about 150 nucleotides, greater than about 200 nucleotides, or greater than about 250 nucleotides. Alternatively, or in addition thereto, the method can include removing DNA from the sample from the subject, for example by contacting the sample with a DNAse.

[0095] The methods of the present disclosure are applicable to a broad range of antiviral therapies. In some aspects, the method includes administering a therapeutic agent selected from an antiviral agent, a vaccine, a vaccine adjuvant, an immune stimulant, an antibody, a toll-like receptor agonist, a cytokine, a chemokine, an interleukin, an oligodeoxynucleotide, a glucan, a type I interferon receptor agonist, a type II interferon receptor agonist, an anti-inflammatory agent, a nuclease, a cell-based therapy, or a combination thereof. In particular aspects, the antiviral agent includes ABT-263, alisporivir, amiodarone, arbidol, bevacizumab, boceprevir, calpain inhibitor II, calpain inhibitor XII, camostat, camphor, carvedilol, cepharanthine, chloroquine, colchicine, corticosteroids, cyclosporine, dactinomycin, dalbavancin, dasatinib, dexamethasone, eflornithine, emetine, emodin, emodin, eplerenone, equilin, famotinine, favilavir, favipiravir, flavipavir, flovoxamine, galdecivir, GC-376, gemcitabine, glycyrrhizin, GS-441524, hexachlorophene, homoharringtonine, hydroxychloroquine, indomethacin, irbesartan, ivermectin, lopinavir, lopinavir, luteolin, mefloquine, memantine, mercaptopurine, mesalazine, monesin, nafamosta, nelfinavir, niclosamide, nitazoxanide, oritavancin, oxymetholone, paroxetine, promethazine, quinacrine, rapamycin, ribavirin, ritonavir, ritonavir, saracatinib, sarilumab, silvestrol, sirolimus, solnatide, teicoplanin, telavancin, tilorone, tocilizumab, toremifene, trametinib, umifenovir, or remdesivir.

[0096] The methods of the present disclosure enable early-phase detection of viral infections, including for subjects that are asymptomatic of an infection. For example, for subjects with pulmonary infections (e.g., SARS-CoV-1 or SARS-CoV-2), the subject can have a chest X-ray (SCR) score of less than about 25, of less than about 24, of less than about 23, of less than about 22, of less than about 21, of less than about 20, of less than about 19, of less than about 18, of less than about 17, of less than about 16, of less than about 15, of less than about 14, of less than about 13, of less than about 12, of less than about 11, of less than about 10, of less than about 9, of less than about 8, of less than about 7, of less than about 6, of less than about 5, of less than about 4, or of less than about 3. In another aspect, the infection is a chronic infection (e.g., a latent chronic infection).

[0097] In some aspects, the modulated expression is expression relative to a control sample from a non-infected subject. As a non-limiting example, the non-infected subject can be ahuman who is confirmed to not have a viral infection, for example through use of a viral panel or a viral PCR test.

[0098] In another embodiment, the present disclosure provides a method of identifying a subject as a likely responder to antiviral therapy that includes: a) detecting in a sample from the subject: i) short viral RNA (svRNA) with a length of about 10-50 nucleotides, ii) modulated expression of 10-50 nucleotide RNA in the subject relative to a control sample, or iii) a combination thereof; and b) classifying the subject: as a likely responder to antiviral therapy based on the svRNA detected in the sample, the modulated expression of the RNA in the subject relative to the control sample, or the combination thereof, thereby identifying the subject as a likely responder to antiviral therapy.

[0099] In one aspect, the detecting includes use of a primer that includes a sequence selected from SEQ ID NOs: 1-11. In another aspect, the svRNA includes a sequence selected from SEQ ID NOs: 12-19. In a further aspect, the RNA in the subject includes a sequence selected from SEQ ID NOs:20-39.

[0100] As non-limiting examples, the control sample can be a sample from a non-responder to the antiviral therapy, a sample from a subject that does not have a viral infection, or a combination thereof. Alternatively, or in addition thereto, the control sample can be a standard generated from the subject (e.g., at a point in time in which the subject does not have a viral infection) or a representative subject.

[0101] In some cases, the viral infection is an infection with a positive-strand RNA virus. In a particular aspect, the viral infection is a coronavirus infection. In a specific aspect, the viral infection is a SARS-CoV-1 or SARS-CoV-2 infection.

[0102] In another aspect, the classifying includes determining the severity of the viral infection. In particular, changes in levels of the svRNAs and / or the RNA from the subject can serve as an indicator to recognize whether a person suffers from a viral infection (e.g., SARS- CoV-2), whether the infection is in an early, intermediate, or advanced stage, and whether the symptoms of the infection are mild, moderate, or severe. As an example, the detecting can include quantifying the svRNA and / or a degree of the modulated expression of the RNA in the subject, wherein the level of svRNA and / or the degree of expression modulation indicates a severity of the infection. The quantifying can include, for example, measuring a count per million of the svRNA and / or RNA in the subject. This concept is illustrated by the data in FIG. IB, which show increases in svRNA over the first 24 hours of SARS-CoV-1 and SARS-CoV- 2 infection. The detecting can also include measuring a change in a level of the svRNA and / orexpression of the RNA in the subject to determine whether a viral infection increased or decreased in severity, for example whether a viral load increased or decreased. Similarly, the classifying can include identifying a subject who does not exhibit symptoms of an infection as having an early-phase viral infection.

[0103] Alternatively, or in addition thereto, the classifying can include determining that the subject has a viral infection.

[0104] Further disclosed herein are compositions and kits for performing a method of the present disclosure, as well as methods of using the composition or kit to detect RNA in a biological sample. For example, in one embodiment, the present disclosure provides a composition with a nucleic acid that includes a sequence selected from SEQ ID NOs: l-l l. In some aspects, the composition includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or 11 sequences selected from SEQ ID NOs: 1-11.

[0105] As a further example, the present disclosure provides a kit that contains a composition of the present disclosure, such as a composition that includes a nucleic acid with a sequence selected from SEQ ID NOs: 1-11, or a composition that includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or 11 sequences selected from SEQ ID NOs: 1-11. As further non-limiting examples, the kit can also include: i) a lysis buffer, ii) a DNAse, iii) a reverse transcriptase, iv) a universal primer, v) a dNTP, vi) a filter, vii) a ligase, viii) an adapter, barcode, or polyadenylation sequence, ix) a multiwell plate, or x) a combination thereof. In particular aspects, two or more wells of the multiwell plate comprise different primers selected from SEQ ID NOs: 1-11.

[0106] As used here the term “lysis buffer” refers to a solution that breaks open cells to release their contents for analysis or experimentation. Lysis buffers contain chemical agents like detergents, inhibitors, and stabilizers. The type of lysis buffer used depends on the proteins or organelles being extracted, and the tissue source. Examples of lysis buffers may include but are not limited to RIPA buffer, NP-40 lysis buffer, SDS lysis buffer, Tris-NaCl-EDTA (TNE) buffer, laemmli sample buffer, CHAPS lysis buffer, Urea lysis buffer, PBS-based lysis buffer, and specialized lysis buffers for specific organelles like nuclear lysis buffer or mitochondrial lysis buffer.

[0107] As used herein the term “deoxyribonuclease” or “DNase” refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. DNases can be used to detect regulatory DNA.

[0108] As used here the term “reverse transcriptase” or “RT” an enzyme that converts RNA into DNA, a process called reverse transcription. RT is a key part of the replication cycle for various viruses.

[0109] As used herein the term “universal primer” refers to a type of DNA primer designed to bind to a conserved region of DNA that is common across a wide range of organisms or species, allowing for amplification of a specific gene sequence from diverse samples within that group, essentially acting as a "generic" primer for multiple related organisms. Universal primers are often used in PCR techniques for microorganism identification or genetic analysis.

[0110] As used herein the term “dNTP” refers to a monomeric unit of DNA that serves as an energy source and building block for DNA replication and maintenance.[OHl] As used herein the term “filter” refers to a device that separates solids from a fluid stream.

[0112] As used herein the term “ligase” refers to an enzyme that joins two molecules together by forming a new chemical bond.

[0113] As used herein the term “adaptor” refers to a short, synthetic DNA fragment used in genetic engineering or a protein that connects signaling proteins. Adapters for genetic engineering are short, double-stranded DNA fragments that are used to join DNA fragments that have been cut with different restriction enzymes. Adapters have different sequences at their ends and are often pre-ligated to the DNA fragment. They are used in techniques like polymerase chain reaction (PCR), next-generation sequencing (NGS), and molecular cloning.

[0114] As used herein the term “barcode” refers to a short DNA sequence that uniquely identifies a species. DNA barcoding is a method of specimen identification using short, standardized segments of DNA. Every species has its own barcode.

[0115] As used herein the term “polyadenylation sequence” or “poly(A) signal” or “PAS” refers to a sequence motif that is a defining feature of eukaryotic protein-coding genes. The PAS is part of a three-step process that forms the 3' ends of most eukaryotic mRNAs: recognition, cleavage, and addition. The added stretch of polyadenosine monophosphate is called the polyadenylation tail (POLYA TAIL).

[0116] As used herein the term “multiwell plate” or “microplate” or “microtiter plate” refers to a flat plate with multiple small wells, essentially acting like a tray of tiny test tubes, used in laboratories to perform multiple simultaneous experiments with small volumes of liquid samples, often in research fields like molecular biology, diagnostics, and cell culture. Multiwell plate allows for efficient analysis of many samples at once.

[0117] In another embodiment, the present disclosure provides a method of detecting infection by a virus in a subject that includes: a) detecting 10-50 nucleotide length short viral RNA (svRNA), modulated expression of 10-50 nucleotide length RNA in the subject, or a combination thereof, by: i) polyadenylating the svRNA, the RNA from the subject, or the combination thereof, thereby generating polyadenylated RNA, ii) reverse transcribing the polyadenylated RNA, thereby generating DNA, and iii) performing quantitative PCR on the DNA, thereby detecting the svRNA, the modulated expression of the RNA in the subject, or the combination thereof; thereby detecting the infection by the virus in the subject.

[0118] In an additional embodiment, the present disclosure provides a method that includes: a) detecting in a sample from a subject: i) RNA that includes a sequence selected from SEQ ID NOs: 12-19 and a length of 10-50 nucleotides, ii) modulated expression of RNA comprising a sequence selected from SEQ ID NOs:20-39 and a length of 10-50 nucleotides, or iii) a combination thereof, thereby detecting infection by a virus in the subject. The method can optionally include administering a therapeutic agent to the subject, thereby treating the infection in the subject.

[0119] In one aspect, the virus is SARS-CoV-1 or SARS-CoV-2. In an additional aspect, the detecting includes the use of a primer that includes a sequence selected from SEQ ID NOs: l- 11.

[0120] In a further embodiment, the present disclosure provides a method of determining the severity of a viral infection in a subject that includes: a) quantifying in a sample from the subject: i) an amount of short viral RNA (svRNA) with a length of about 10-50 nucleotides, ii) a degree of modulated expression of 10-50 nucleotide RNA in the subject relative to a control sample, or iii) a combination thereof; and b) determining the severity of the viral infection based on the svRNA detected in the sample, the modulated expression of the RNA in the subject relative to the control sample, or the combination thereof, thereby determining the severity of the viral infection in the subject.

[0121] In some aspects, the quantifying includes determining a change in the amount of the svRNA in the sample, a change in a degree of the modulated expression of the 10-50 nucleotide RNA in the subject relative to the control sample, or a combination thereof.

[0122] Presented below are examples discussing detection of svRNA derived from SARS- CoV-1 and SARS-CoV-2 for determining infection status contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typicalof those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.EXAMPLES EXAMPLE 1 Materials and Methods

[0123] Raw small RNA-seq datasets

[0124] Raw fastq files were downloaded from the GEO database (accession number GSE148729). The small RNA sequencing dataset included six controls (SRR11550015, SRR11550016, SRR11550017, SRR11550018, SRR11550031, SRR11550032) and twelve samples from cells infected with either SARS-CoV-1 (SRR11550019, SRR11550020, SRR11550021, SRR11550022, SRR11550023, SRR11550024) or SARS-CoV-2 (SRR11550025, SRR11550026, SRR11550027, SRR11550028, SRR11550029, SRR11550030). The total RNA sequencing and mRNA sequencing datasets from the same mock and SARS-CoV-1 / 2 infected cells were used to evaluate the origin of certain small RNA- seq reads.

[0125] Bioinformatics Analysis

[0126] Raw small RNA-seq reads were trimmed of poly(A) tails and adapter sequences including the first 3 nucleotides using cutadapt software (cutadapt-u 3 input.fastq \ cutadapt -a AAAAAAAA -o output. fastq). Reads shorter than 15 nucleotides were discarded. The trimmed and size-selected reads were further aligned to either SARS-CoV-1 or SARS-CoV-2 reference genomes (NC_004718.3 and NC_045512.2, respectively) with bowtie, allowing no mismatches (- v 0 option). The reads derived from forward (+) and reverse (-) strands were differentiated using —norc and —nofw parameters, respectively, during alignment with bowtie. Reads exclusively aligned to either SARS-CoV-1 or SARS-CoV-2 were extracted using a combination of samtools, bam2fastx and bowtie packages. Sorted and indexed BAM files were generated using samtools, and the alignments to the reference viral genomes were visualized in IGV. Small RNA-seq datasets (filtered from all SARS-CoV-1 / 2 reads) were mapped to human references in a sequential manner; specifically, trimmed and size-selected reads were first mapped to RNA species having low sequence complexity and / or a large number of repeats, including rRNA, tRNA, RN7S, snRNA, snoRNA / scaRNA, vault RNA, RNY (custom-curated references from NCBI RefSeq and GENCODE) and the mitochondrial chromosome, from GROG 8. All reads that did not map to the aforementioned RNAs were sequentially aligned to mature miRNA(miRBase 22 release), pre-miRNA (miRBase 22 release), protein-coding mRNA transcripts and long non-coding RNAs (custom-curated references from GENCODE v32). Remaining unmapped reads were aligned to the remaining transcriptome (GENCODE v32), containing mostly pseudogenes and non-protein-coding parts of mRNA transcripts. Finally, reads not mapping to the human transcriptome were aligned to the human primary GROG 8 assembly, corresponding to reads derived from introns and intergenic regions. The numbers of reads mapped to each RNA reference type were extracted using eXpress software based on a previous publication [Roberts and Pachter, 2013], The differential gene expression analysis of both small RNA-seq and mRNA-seq was done using R / B ioconductor packages edgeR and limma.

[0127] RT-qPCR validation of svRNA from independent infection experiments

[0128] Human lung adenocarcinoma (A549) cells overexpressing the ACE2 receptor were seeded at 1 million cells per well in a 6-well plate. Three wells were infected with at a multiplicity of infection (MOI) of 5.0 tissue culture infectious units (TCID50) per cell with SARS-CoV-2 / USA / DC-HP00007 / 2020 (GenBank: MT509464.1) virus, and three wells remained uninfected. RNA was isolated from cells 24h after infection using the miRNeasy micro kit (Qiagen, Hilden, Germany) and Zymo-Spin IIICG columns. RNA was eluted in 50 pl RNase-free water, and RNA concentration was measured by NanoDrop One UV-Vis spectrophotometer (ThermoFisher). cDNA was prepared by miScript RT II cDNA kit (Qiagen) according to manufacturer’s instructions, using HiFlex buffer and 4 pg RNA input. No-RT controls were included to confirm the absence of co-isolated cellular DNA. Next, cDNA was diluted tenfold and used in qPCR; 2 pl of diluted cDNA template was mixed with 4 pl QuantiTect SYBR Green PCR Master Mix (Qiagen), 100 nM forward primers (see TABLE 1 for sequences) and 100 nM miScript universal reverse primer. After an initial polymerase activation step (15 minutes at 95°C), 50 cycles of the following program were run: 15 sec at 94C, 30 sec at 55C, 30 sec at 70C, followed by melting curve analysis on a BioRad CFX96 machine. A complete listing of differentially expressed human transcripts in small RNA-seq datasets is available at< biorxiv.org / content / 10.1101 / 2022.12.27.522023v2.supplementary-material>.

[0129] TABLE 1EXAMPLE 2Small RNAs Derived from SARS-CoV-1 and SARS-CoV-2

[0130] This example explores the repertoire of short viral RNAs in SARS-CoV-1 and SARS- CoV-2. Publicly available small RNA-seq datasets from Calu-3 cells infected with SARS- CoV-1 or SARS-CoV-2 (FIG. 1A) were analyzed for svRNAs. These data sets included raw sequencing data generated by a library preparation kit not biased towards 5’-Phosphate / 3’-OH small RNAs. Noting that viral short RNA fragments might be used for COVID-19 diagnosis and monitoring using qPCR-based assays, small RNA-seq reads from both mock and infected cells were trimmed from adapters, size-selected and aligned to the corresponding viral reference genome. Next, the count-per-million (CPM) values of reads mapped to each reference were calculated. Infection with either virus caused robust accumulation of short RNA fragments derived from both forward and reverse strands of viral RNA genomes 4, 12 and 24h post infection in Calu-3 cells (FIG. IB). Furthermore, the number of reads mapped to the forward viral RNA strands was approximately 20-fold higher than those aligned to the reverse strands (FIG. IB) Perfect-match viral sequences in the small RNA fractions of Calu-3 cells 24 h post-infection were 2.83% (2.68% forward + 0.15% reverse) of the total for SARS-CoV- 1 and 1.51% (1.42% forward + 0.09% reverse) for SARS-CoV-2 (FIG. 1C). This almost 2- fold difference could be explained by significantly higher cytotoxicity of SARS-CoV-2. As could be expected, some SARS-CoV-1 small RNA reads aligned to the SARS-CoV-2 reference genome and vice versa due to the inherent similarity of the genomes (FIG. 1C).EXAMPLE 3Dissection and Validation of SARS-CoV-l / 2-Derived Short RNAs

[0131] This example overviews the distribution pattern of short viral RNA (svRNA) reads and virus-specific fragments mapping to discrete locations (peaks). For this purpose, reads aligning exclusively to either the SARS-CoV-1 or SARS-CoV-2 genome (forward or reverse strands) were extracted and overlapping fragments were discarded. As evident from the genome browser maps, multiple reads aligned discretely to single positions within SARS-CoV- 1 or SARS-CoV-2 genomes (FIGs 2A-2C, 6, 7).

[0132] To assess the presence of five selected svRNAs annotated as 245 IF, 3178F, 29096F, 23339R and 2323R, along with two RNA sequences with high local coverage - 29364F and 29841F (FIG. 2A-2C), in a different infection setting, RNA from infected and uninfected A549 ACE2-cells (FIG. 3) was analyzed. In FIG. 2C, the symbol indicates the number of times a given sequence is present in the corresponding dataset as counted using the grep command in Unix Bash / Shell. Polyadenylation and reverse transcription were followed by real-time quantitative PCR (FIG. 3A, FIG. 3B). For all five putative forward- strand svRNAs (245 IF, 3178F, 29096F, 29364F and 29841F), signal in infected cells was greatly increased over background (FIG. 3C). In contrast, reverse-strand fragments 23339R and 2323R were not detected over background (FIG. 3C). Importantly, dependance on the addition of poly(A) tails to the 3’ end of small RNAs supports the notion that detected svRNAs were amplified from bona- fide small RNA templates rather than full-length viral RNAs. Three negative control primers that annealed to SARS-CoV-2 genomic regions with fewer than 30 counts in the coverage plots were also tested (FIG. 3C). For two, signal was similar in infected and uninfected cells (FIG. 3C). Interestingly, the third negative control (nc 14008) appeared to be induced in infected cells. However, amplification occurred even later than for the putative SARS-CoV-2 small RNA with the latest amplification (Cq 25.5 vs 23.0) (FIG. 3C). These data thus suggest that the SARS- CoV-2 genome may encode small RNA fragments of a discrete size.EXAMPLE 4Read Counts of SARS-CoV-2 Viral miRNAs Versus Other Short RNAs

[0133] SARS-CoV-2 expresses endogenous miRNAs such as CoV2-miR-O7a and its isoform CoV2-miR-O7a.2, encoded in the SARS-CoV-2 ORF7, and CvmiR-5 encoded in ORF la. However, in the SRR11550027 and SRR11550028 datasets (small RNA-seq of cells24h post-infection) only a small number of these viral miRNA sequences were found. Specifically, the top three short viral RNAs identified in the present study were up to 100- to 1000-fold more abundant than previously reported viral miRNA reads in the same samples(TABLE 2, FIG. 2C)

[0134] TABLE 2EXAMPLE 5Host Small RNA Expression In Control and Infected Cells

[0135] In their original publication, Wyler et al restricted differential expression analysis of small RNA-seq data in control and infected cells to miRNA as well as vault RNA [Wyler et al, 2022], These analyses were expanded herein to the whole-transcriptome level by mapping total reads to a custom-curated human reference transcriptome in a sequential manner as described in Materials and Methods (FIG. 4A). Subsequently, differential expression analysis wasperformed for mock-infected controls versus SARS-CoV-1 and SARS-CoV-2-infected cells at 24h post-infection. Significantly more small RNA transcripts were differentially expressed in SARS-CoV-2 versus SARS-CoV-1 infection (FIG. 4B). Specifically, of 14747 transcripts remaining after low-expression filtering, only 12 up- and 0 down-regulated sequences were identified in SARS-CoV-1 infection, compared with 268 up-and 120 down-regulated sequences in SARS-CoV-2 infection (FIG. 4C). Only 7 differentially expressed transcripts overlapped between SARS-CoV-1 and SARS-CoV-2 infected cells (FIG. 4B). Interestingly, the percentage of reads mapped to mature miRNAs and pre-miRNAs combined was significantly lower than the proportion of reads mapped to other RNA classes (FIG. 4D). Thus, only 1 and 45 miRNAs / premiRNAs showed significant (adj. p-values < 0.05) differential expression in SARS-CoV-1 and SARS-CoV-2 infection, respectively. While hsa- miR-155-3p was markedly upregulated in both SARS-CoV-1 and SARS-CoV-2 treated cells (LFC 4.0 and 2.7, respectively), the majority of miRNAs / premiRNAs showed relatively subtle changes 24h after SARS-CoV-2 infection (-2 < LFC < 2). In contrast, fragments of proteincoding mRNAs and IncRNAs predominated among the differentially expressed transcripts (FIG. 4D). Finally, SARS-CoV-2 infection induced a strong upregulation of short RNA fragments deriving from 3’ - and 5’ - termini of full-length Y-RNAs (LFC 3.0), and to a lesser extent vault RNA (LFC 1.61), as well as tRNA (LFC 1.25) (FIG. 4D). On the levels of individual tRNAs, however, a more profound upregulation was observed, with LFC values between 2.94 - 5.06 for the top 10 differentially expressed tRNAs (TABLES 4-6).

[0136] TABLE 4

[0137] TABLE 5

[0138] TABLE 6

[0139] Interestingly, the size distribution of reads mapped to Y-RNAs, vault RNAs and tRNAs was noticeably different in SARS-CoV-2 infected cells as compared with mock- infected controls, indicating that the infection induced generation of a distinct set of short RNAs from the aforementioned sncRNAs (FIG. 8A-8C). TABLE 7 provides the top 5 overrepresented RNY-, RNA-, and tRNA reads in SARS-CoV-2 infected Calu-3 cells from the data shown in FIG. 8A-8C. The full-length Y-RNAs, vault RNAs and tRNAs were not found in the small RNA-seq dataset, indicating that the methods applied for RNA isolation and library preparation were highly biased to capturing RNAs of shorter sizes (FIG. 8A-8C). In addition, RNY, vault RNA and tRNA reads mapped predominantly to 3’- and 5’- termini of the parental transcripts (FIG. 9-11).

[0140] TABLE 7

[0141] Finally, a number of miscellaneous Y-RNA transcripts, mapped to various Y-RNA pseudogenes scattered across human genome, were found in the small RNA datasets from SARS-CoV-2 infected cells (FIG. 12, TABLE 8). In FIG. 12, all reads mapped to the main four RNY genes were removed before aligning to GENCODE v.41 RNY references. Many of these minor Y-RNA sequences have very marginal presence in the uninfected cells but accumulated robustly within 24h of SARS-CoV-2 infection (TABLES 4-6).

[0142] TABLE 8EXAMPLE 6Short RNAs Deriving from SARS-CoV-1 / 2 Also Align to Various hg38 Sequences

[0143] This example overviews whether identified SARS-CoV-1 and SARS-CoV-2 short viral RNAs have similarities with human sequences, and thus could potentially target host transcripts. For this purpose, viral RNA reads extracted from the investigated small RNA datasets were aligned to human transcriptome references and subsequently genomic DNA with 0 mismatch tolerance (FIG. 5A). Mapping was done to forward (bowtie —norc) and reverse (bowtie —nojw) orientations separately, identifying viral RNAs aligning to hg38 transcriptome in both sense and antisense orientation (FIG. 5A). The reads that did not align to human transcriptome references were further mapped to human genomic DNA (without strandspecificity), which allowed reads to be separated based on mapping to introns and intergenic regions exclusively. Interestingly, 27.23% - 30.87% (depending on mapping) of total SARS- CoV-1 and 16.49% 16.81% of total SARS-CoV-2 reads aligned to human introns and intergenic regions (FIG. 5B) Furthermore, a significant proportion of total viral reads were mapped to human protein-coding and IncRNA transcriptome. It should be noted that the vast majority of SARS-CoV-1 and SARS-CoV-2 reads that aligned to human sequences were 15-18 nt in size (FIG. 5C, 5D) and mapped to a single location within certain mRNA or IncRNA genes.EXAMPLE 7 qPCR Analysis for the 2451F, 3178F, 29096F short viral RNAs in Bronchial Fluid

[0144] Two independent cDNA synthesis (run_181837 and run_l 84229) were performed as duplicates in a qPCR plate. 50 pg of synthetic cel-miR-39 were spiked in before RNA isolation using Trisol LS reagent and used for normalization of RNA isolation efficacy. C1-C7 depicts RNA isolated from 400 uL bronchial fluid collected from individuals diagnosed with SARS- CoV-2 during acute phase. K saliva depicts RNA isolated from 400 uL saliva sample from a SARS-CoV-2 negative individual without any sign of the disease. Input for the RT reaction was prepared by dissolving 50 pg or 5 pg of synthetic 22nt cel-miR-39 in 50ul of RNAase free water. RNAase free water was used as input for RT reaction (H2O RT). RNAase free water was also used as input for qPCR reaction (H2O qPCR). F29096 indicates forward qPCR primer for peak labelled as 29096F in FIG. 3C; the reverse primer is universal. F2451 indicates forward qPCR primer for peak labelled as 245 IF in FIG. 3C; reverse primer is universal. F3178 indicates forward qPCR primer for peak labelled as 3178F in FIG. 3C; reverse primer is universal. Fc39 indicates forward qPCR primer specific for synthetic spiked-in cel-miR-39; reverse primer is universal. c39 bioliqx indicates forward qPCR primer and reverse qPCR primers specific for synthetic cel-miR-39 from BioLiqX qPCR QC kit (HD Biolabs).

[0145] Two out of the 5 bronchial samples (C6 and C7 - labeled as "clot") were visibly more concentrated as compared to the others samples which appeared more transparent. Clot samples likely contained dead cells from the infected lung / bronchial epithelia. As illustrated in FIG. 13 A-13F and int Table 9, in those samples, for all three forward-strand svRNAs (245 IF, 3178F, and 29096F), signal was greatly increased as compared to the control, reflecting that the presence of SARS-CoV-2 short viral RNAs was significantly higher in these samples. These results indicate that pure bronchial fluid likely does not carry 245 IF, 3178F, and 29096F svRNAs. TheC6 and C7 samples likely contained cells from the infected lung / bronchial epithelia and thus showed high content of the svRNAs. (FIG. 13A-13F).

[0146] Futures analysis will include the assessment of the detection of the svRNAs in blood plasma smples.

[0147] TABLE 9

[0148] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What Is Claimed Is:

1. A method comprising: a) detecting in a sample from a subject: i) 10-50 nucleotide length short viral RNA (svRNA), ii) modulated expression of 10-50 nucleotide length RNA in the subject, or iii) a combination thereof, thereby detecting infection by a virus in the subject.

2. The method of claim 1, further comprising administering a therapeutic agent to the subj ect.

3. The method of claim 1, wherein the virus is a positive-strand RNA virus.

4. The method of claim 3, wherein the virus is a coronavirus.

5. The method of claim 4, wherein the virus is SARS-CoV-1 or SARS-CoV-2.

6. The method of claim 3, wherein the svRNA maps to a positive strand of a genome of the virus.

7. The method of claim 1, wherein the svRNA maps to the human genome.

8. The method of claim 1, wherein the detecting comprises measuring a prevalence of at least about 1000 counts per million, at least about 2000 counts per million, at least about 3000 counts per million, at least about 4000 counts per million, at least about 5000 counts per million, at least about 6000 counts per million, at least about 7000 counts per million, at least about 8000 counts per million, at least about 9000 counts per million, at least about 10000 counts per million, at least about 12000 counts per million, at least about 15000 counts per million, at least about 20000 counts per million, or at least about 25000 counts per million of the svRNA.

9. The method of claim 1, wherein the short viral RNA comprises one or more sequences selected from SEQ ID NOs: 12-19.

10. The method of claim 1, wherein the RNA from the subject comprises a fragment of mRNA, IncRNA, vault RNA, tRNA, or Y-RNA.

11. The method of claim 10, wherein the RNA from the subject comprises one or more sequences selected from SEQ ID NOs:20-39.

12. The method of claim 1, wherein the svRNA, the RNA from the subject, or the combination thereof comprises between 2 and 10 sequences, between 2 and 20 sequences, between 2 and 30 sequences, between 2 and 40 sequences, between 2 and 50 sequences, between 2 and 75 sequences, between 2 and 100 sequences, between 2 and 200 sequences, between 2 and 500 sequences, between 5 and 10 sequences, between 5 and 20 sequences, between 5 and 40 sequences, between 5 and 50 sequences, between 5 and 75 sequences, between 5 and 100 sequence, between 5 and 200 sequences, between 5 and 500 sequences, between 10 and 20 sequences, between 10 and 30 sequences, between 10 and 40 sequences, between 10 and 50 sequences, between 10 and 75 sequences, between 10 and 100 sequences, between 10 and 200 sequences, between 10 and 500 sequences, between 20 and 30 sequences, between 20 and 40 sequences, between 20 and 50 sequences, between 20 and 100 sequences, between 20 and 200 sequences, between 20 and 500 sequences, between 30 and 50 sequences, between 30 and 100 sequences, between 30 and 200 sequences, between 30 and 500 sequences, between 50 and 100 sequences, between 50 and 200 sequences, between 50 and 500 sequences, between 100 and 200 sequences, between 100 and 500 sequences, or between 200 and 500 sequences.

13. The method of claim 1, wherein the svRNA, the RNA from the subject, or the combination thereof comprises a length of about 15-26 nucleotides.

14. The method of claim 1, wherein the svRNA, the RNA from the subject, or the combination thereof comprises multiple RNA biotypes.

15. The method of claim 1, wherein an RNA molecule of the svRNA, the RNA from the subject, or the combination thereof does not comprise a 5 ’-phosphate, a 3 ’-hydroxyl, or a combination thereof.

16. The method of claim 1, wherein the sample comprises a cell lysate or a bronchial fluid.

17. The method of claim 1, wherein the detecting comprises reverse transcription, polymerase chain reaction (PCR), real-time PCR (rt-PCR), quantitative PCR (qPCR), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, rolling circle amplification, in-vitro transcription (IVT), strand displacement amplification, fluorescence in situ hybridization (FISH), transcription-mediated amplification(TMA), Eberwine amplification, microarray detection, electrophoresis, high pressure liquid chromatography (HPLC), affinity chromatography, CRISPR-based detection, or a combination thereof.

18. The method of claim 1, wherein the detecting comprises ligating an adapter, a barcode, or a polyadenylation sequence to the svRNA, the RNA from the subject, or the combination thereof.

19. The method of claim 1, wherein the detecting comprises use of a primer comprising a sequence selected from SEQ ID NOs: l-l l.

20. The method of claim 1, wherein the detecting comprises reverse transcription of the svRNA, the RNA from the subject, or the combination thereof to generate DNA, and detecting a sequence of the DNA.

21. The method of claim 1, wherein the detecting comprises separating the svRNA, the RNA from the subject, or the combination thereof from the sample from the subject.

22. The method of claim 1, wherein the detecting comprises separating the svRNA, the RNA from the subject, or the combination thereof from nucleic acids with lengths greater than about 50 nucleotides, greater than about 75 nucleotides, greater than about 100 nucleotides, greater than about 150 nucleotides, greater than about 200 nucleotides, or greater than about 250 nucleotides.

23. The method of claim 22, wherein the separating comprises filtration, extraction, precipitation, or a combination thereof.

24. The method of claim 1, wherein the detecting comprises: i) polyadenylating the svRNA, the RNA from the subject, or the combination thereof, thereby generating polyadenylated RNA, ii) reverse transcription of the polyadenylated RNA, thereby generating DNA, and iii) quantitative PCR of the DNA.

25. The method of claim 1, further comprising administering a therapeutic agent selected from an antiviral agent, a vaccine, a vaccine adjuvant, an immune stimulant, an antibody, a toll-like receptor agonist, a cytokine, a chemokine, an interleukin, an oligodeoxynucleotide, aglucan, a type I interferon receptor agonist, a type II interferon receptor agonist, an antiinflammatory agent, a nuclease, a cellular therapy, or a combination thereof.

26. The method of claim 25, wherein the antiviral agent comprises ABT-263, alisporivir, amiodarone, arbidol, bevacizumab, boceprevir, calpain inhibitor II, calpain inhibitor XII, camostat, camphor, carvedilol, cepharanthine, chloroquine, colchicine, corticosteroids, cyclosporine, dactinomycin, dalbavancin, dasatinib, dexamethasone, eflornithine, emetine, emodin, emodin, eplerenone, equilin, famotinine, favilavir, favipiravir, flavipavir, flovoxamine, galdecivir, GC-376, gemcitabine, glycyrrhizin, GS-441524, hexachlorophene, homoharringtonine, hydroxychloroquine, indomethacin, irbesartan, ivermectin, lopinavir, lopinavir, luteolin, mefloquine, memantine, mercaptopurine, mesalazine, monesin, nafamosta, nelfinavir, niclosamide, nitazoxanide, oritavancin, oxymetholone, paroxetine, promethazine, quinacrine, rapamycin, ribavirin, ritonavir, ritonavir, saracatinib, sarilumab, silvestrol, sirolimus, solnatide, teicoplanin, telavancin, tilorone, tocilizumab, toremifene, trametinib, umifenovir, or remdesivir.

27. The method of claim 1, wherein the subject is asymptomatic of the infection.

28. The method of claim 1, wherein the subject received a chest X-ray (SCR) score of less than about 25, of less than about 20, of less than about 15, of less than about 12, of less than about 10, of less than about 8, of less than about 6, of less than about 5, of less than about 4, or of less than about 3.

29. The method of claim 1, wherein the infection is a chronic infection.

30. The method of claim 1, wherein the modulated expression is expression relative to a control sample from a non-infected subject.

31. A method of identifying a subject as a likely responder to antiviral therapy comprising: a) detecting in a sample from the subject: i) short viral RNA (svRNA) with a length of about 10-50 nucleotides, ii) modulated expression of 10-50 nucleotide RNA in the subject relative to a control sample, or iii) a combination thereof; and b) classifying the subj ect as a likely responder to antiviral therapy based on the svRNA detected in the sample, the modulated expression of the RNA in the subject relative to the control samplethereby identifying the subject as a likely responder to antiviral therapy.

32. The method of claim 31, wherein the detecting comprises use of a primer comprising a sequence selected from SEQ ID NOs: l-l l.

33. The method of claim 31, wherein the svRNA comprises a sequence selected from SEQ ID NOs: 12-19.

34. The method of claim 31, wherein the RNA in the subject comprises a sequence selected from SEQ ID NOs:20-39.

35. The method of claim 31, wherein the control sample is a sample from a non-responder to the antiviral therapy, a sample from a subject that does not have a viral infection, or a combination thereof.

36. The method of claim 31 , wherein the classifying comprises determining that the subj ect has a viral infection.

37. The method of claim 36, wherein the viral infection is infection with a positive-strand RNA virus.

38. The method of claim 37, wherein the viral infection is a coronavirus infection.

39. The method of claim 37, wherein the viral infection is a SARS-CoV-1 or SARS-CoV- 2 infection.

40. The method of claim 36, wherein the classifying comprises determining the severity of the viral infection.

41. The method of claim 31, wherein the detecting comprises quantifying the svRNA, quantifying a degree of the modulated expression of the RNA in the subject, or the combination thereof in the sample from the subject.

42. A kit for performing the method of claim 31.

43. A composition comprising a nucleic acid comprising a sequence selected from SEQ ID NOs:l-l l.

44. The composition of claim 43, comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or 11 sequences selected from SEQ ID NOs: 1- 11.

45. A kit comprising the composition of claim 43.

46. The kit of claim 45, further comprising: i) a lysis buffer, ii) a DNAse, iii) a reverse transcriptase, iv) a universal primer, v) a dNTP, vi) a filter, vii) a ligase, viii) an adapter, barcode, or polyadenylation sequence, ix) a multiwell plate, or x) a combination thereof.

47. The kit of claim 37, wherein two or more wells of the multiwell plate comprise different primers selected from SEQ ID NOs: l-l l.

48. A method for using the composition of claim 34 or the kit of claim 36, comprising detecting svRNA in a biological sample.

49. A method of detecting infection by a virus in a subject comprising: a) detecting 10-50 nucleotide length short viral RNA (svRNA), modulated expression of 10-50 nucleotide length RNA in the subject, or a combination thereof, comprising: i) polyadenylating the svRNA, the RNA from the subject, or the combination thereof, thereby generating polyadenylated RNA, ii) reverse transcribing the polyadenylated RNA, thereby generating DNA, and iii) performing quantitative PCR on the DNA, thereby detecting the svRNA, the modulated expression of the RNA in the subject, or the combination thereof; thereby detecting the infection by the virus in the subject.

50. A method comprising: a) detecting in a sample from a subject: i) RNA comprising a sequence selected from SEQ ID NOs: 12-19 and a length of 10-50 nucleotides,ii) modulated expression of RNA comprising a sequence selected from SEQ ID NOs:20-39 and a length of 10-50 nucleotides, or iii) a combination thereof, thereby detecting infection by a virus in the subject.

51. The method of claim 50, further comprising administering a therapeutic agent to the subj ect.

52. The method of claim 50, wherein the virus is SARS-CoV-1 or SARS-CoV-2.

53. The method of claim 50, wherein the detecting comprises use of a primer comprising a sequence selected from SEQ ID NOs: l-l l.

54. A method of determining the severity of a viral infection in a subject comprising: a) quantifying in a sample from the subject: i) an amount of short viral RNA (svRNA) with a length of about 10- 50 nucleotides, ii) a degree of modulated expression of 10-50 nucleotide RNA in the subject relative to a control sample, or iii) a combination thereof; and b) determining the severity of the viral infection based on the svRNA detected in the sample, the modulated expression of the RNA in the subject relative to the control sample, thereby determining the severity of the viral infection in the subject.

55. The method of claim 54, wherein the quantifying comprises determining a change in the amount of the svRNA in the sample, a change in a degree of the modulated expression of the 10-50 nucleotide RNA in the subject relative to the control sample, or a combination thereof.

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