A combination of biomarkers for assessing the severity of viremia in Chikungunya virus patients and their applications

Abstract

This application relates to the field of biomedical detection technology, specifically to a combination of biomarkers for assessing the severity of viremia in Chikungunya virus patients and its application. The combination of biomarkers for assessing the severity of viremia in Chikungunya virus patients includes one or more of the following biomarkers: TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1. The biomarker combination provided in this application can objectively and quantitatively reflect the activity of viral replication and the strength of the immune response in the patient, thereby achieving accurate assessment of the severity of viremia. This solves the problem in existing technologies that mainly rely on qualitative or semi-quantitative detection of viral nucleic acids and lack assessment methods closely related to the host's pathological state.

Description

Technical Field

[0001] This application relates to the field of biomedical detection technology, and in particular to a combination of biomarkers for assessing the severity of viremia in patients with Chikungunya virus and their application. Background Technology

[0002] Chikungunya fever is an acute mosquito-borne infectious disease caused by Chikungunya virus (CHIKV), clinically manifested as acute fever, rash, and symmetrical severe polyarthritis. The virus is primarily transmitted by Aedes aegypti and Aedes albopictus mosquitoes, and approximately 2.8 billion people are at risk of infection. About 50% of infected individuals may develop chronic arthritis, with symptoms lasting for years, severely impacting their quality of life. Existing biomarkers primarily target the virus itself (nucleic acid or protein) and cannot reflect the host's immune response to viral infection. Therefore, there is an urgent need to develop a set of host-derived biomarkers that can specifically assess the severity of viremia in CHIKV-infected individuals. Summary of the Invention

[0003] The main objective of this application is to propose a combination of biomarkers for assessing the severity of viremia in patients with Chikungunya virus and their application, aiming to solve the technical problem of the lack of host-derived biomarkers in the prior art that can specifically assess the severity of viremia in Chikungunya virus-infected individuals.

[0004] To achieve the above objectives, in a first aspect, this application proposes a combination of biomarkers for assessing the severity of viremia in patients with Chikungunya virus, comprising one or more of the following biomarkers: TAP1, ISG15, CMPK2, GBP4, OTOF, SIGLEC1.

[0005] In some embodiments, the biomarker further includes CXCL10.

[0006] Secondly, this application also provides a reagent for detecting the biomarkers proposed in this application, said reagent comprising biomolecules that specifically hybridize with the biomarkers proposed in this application or their expression products.

[0007] In some embodiments, the biomolecule includes one or more of primers, probes, and antibodies.

[0008] In some embodiments, the reagents include those for preparing RNA from the biomarker combinations proposed in this application into high-throughput sequencing libraries.

[0009] Thirdly, this application also proposes the use of the combination of biomarkers and / or reagents proposed in this application in the preparation of a kit for assessing the severity of viremia in patients with Chikungunya virus.

[0010] In some embodiments, the test sample of the kit includes one or more of plasma, serum, and whole blood.

[0011] Fourthly, this application also proposes a detection product comprising the combination of biomarkers proposed in this application and / or the reagents proposed in this application.

[0012] In some implementations, the detection product includes one or more chips.

[0013] Fifthly, this application also proposes a method for assessing the severity of viremia in patients with Chikungunya virus, comprising the following steps: detecting the expression level of a combination of biomarkers provided in this application in a biological sample from the patient; and assessing the severity of viremia based on the expression level.

[0014] In some embodiments, the method for detecting the expression level includes: extracting RNA from the biological sample; reverse transcribing the RNA to obtain cDNA; and detecting the mRNA expression level of the combination of biomarkers provided in this application in the cDNA.

[0015] This application identifies seven biomarkers—CXCL10, TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1—through screening, providing a technical solution for directly and accurately assessing the severity of viremia in patients infected with chikungunya virus. Since the expression levels of these biomarkers have been shown to be strongly positively correlated with viral load in patient plasma and are almost undetectable in healthy individuals, detecting their expression levels can objectively and quantitatively reflect the activity of viral replication and the strength of the immune response in patients. This enables precise assessment of the severity of viremia, thus solving the problem of existing technologies that primarily rely on qualitative or semi-quantitative detection of viral nucleic acids and lack assessment methods closely related to the host's pathological state.

[0016] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0018] Figure 1 A correlation analysis diagram of the FPKM values ​​of six host mRNAs in plasma and the viral genome provided for embodiments of this application; Figure 2 A correlation analysis diagram of the relative qPCR quantification values ​​of six host mRNAs in plasma and the FPKM values ​​of the viral genome provided in the embodiments of this application; Figure 3 A graph showing the difference in relative expression levels of six mRNAs in the plasma of the normal group and the infected group, provided in the embodiments of this application; Figure 4 A differential expression analysis diagram of 120 immune-related genes in plasma protein array data provided in the embodiments of this application; Figure 5 A differential expression analysis diagram of transcripts of 120 immune factor-related genes from PBMC cell scRNA-seq data provided in this application embodiment; Figure 6 An analysis diagram of the expression levels of immune-related factors showing differential expression in some plasma protein arrays provided in the embodiments of this application; Figure 7 This is a graph showing the differential expression analysis of plasma CXCL10 concentration (pg / mL) between the infected group and the normal group provided in the embodiments of this application.

[0019] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0021] It should be noted that if the embodiments of this application involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0022] Furthermore, if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application.

[0023] Chikungunya virus infection often presents with viremia during the acute phase, and viral load is closely related to disease severity and clinical prognosis. Currently, clinical assessment of viremia severity primarily relies on direct quantitative detection of viral nucleic acid. However, viral load alone cannot fully reflect the degree of activation of the host's immune system or the intensity of the immune response associated with the pathological state. Therefore, there is an urgent need to discover biomarkers that can directly and objectively reflect the correlation between viral replication activity and host immune activation status, in order to provide a more comprehensive tool for disease assessment.

[0024] To address the aforementioned issues, this application proposes a combination of biomarkers for assessing the severity of viremia in Chikungunya virus patients. This combination includes one or more of the following biomarkers: TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1.

[0025] In some implementations, the biomarker combination used to assess the severity of viremia in Chikungunya virus patients also includes CXCL10.

[0026] CXCL10 is a plasma cytokine, while TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1 are mRNAs in plasma.

[0027] The aforementioned biomarkers originate from the host's immune response to chikungunya virus infection, particularly in peripheral blood mononuclear cells, and their expression is closely related to the widespread activation of the interferon pathway. By detecting the expression levels of these biomarkers in patient plasma and other body fluids, the viral load in the patient can be indirectly but highly correlated, thereby enabling the assessment of the severity of viremia. The mechanism is that when chikungunya virus infection occurs, viral replication activates the host's innate immune response, especially the type I interferon pathway, which in turn induces the expression of a large number of interferon-stimulating genes (ISGs). Genes such as TAP1, ISG15, CMPK2, and GBP4 are typical ISGs, and their expression levels are positively correlated with the intensity of viral stimulation. SIGLEC1 is mainly expressed in monocyte / macrophage cell lines, and its upregulation reflects the activation state of myeloid cells; changes in OTOF expression may be related to functional changes such as immune cell vesicle secretion. Plasma CXCL10 cytokine may be related to symptoms such as joint inflammation in patients. Therefore, the expression profile of the above biomarker combination can comprehensively reflect the degree of virus-induced immune activation and is strongly correlated with the severity of viremia.

[0028] In some implementations, the biomarker combination includes all seven biomarkers: CXCL10 (plasma cytokine), TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1 (plasma mRNA). Using this complete biomarker combination for assessment allows for a more comprehensive and stable capture of the host's immune response to viremia from multiple dimensions (such as broad interferon response and myeloid-specific activation), thereby improving the accuracy and robustness of the assessment. Of course, depending on the actual testing conditions and needs, any two, three, four, or five biomarkers from this combination can be selected for combined detection, such as a combination of TAP1 and ISG15, or a combination of ISG15, CMPK2, and GBP4. Detecting the expression trends of subsets can also reflect the state of viremia to some extent.

[0029] This application also provides a reagent for detecting the above-mentioned combination of biomarkers. The reagent comprises a biomolecule that specifically hybridizes with the seven biomarkers CXCL10, TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1, or their expression products (such as mRNA or protein). Specific hybridization means that the biomolecule can selectively recognize and bind to the target biomarker or its expression product through base pairing or antigen-antibody binding.

[0030] Specifically, biomolecules can include one or more of primers, probes, and antibodies. For example, specific primer pairs and corresponding fluorescent probes targeting TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1 can be designed for real-time quantitative polymerase chain reaction (PCR) detection, as well as an enzyme-linked immunosorbent assay (ELISA) kit targeting CXCL10. Monoclonal or polyclonal antibodies against these six proteins can also be prepared for ELISA or protein chip detection. The sequences or structures of primers, probes, or antibodies can be designed and prepared according to techniques known in the art to ensure high specificity and affinity for their respective targets.

[0031] In some embodiments, the reagent may also include reagents for preparing RNA from these six biomarkers into high-throughput sequencing libraries. For example, the kit may contain reagents for total RNA extraction from plasma, reverse transcription primers with specific barcodes (sequences targeting TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1), enzymes and buffers for cDNA library construction and amplification, etc. By using this reagent, target transcripts in the sample can be enriched and converted into libraries suitable for next-generation sequencing platforms, thereby enabling absolute quantitative analysis of the expression levels of multiple biomarkers.

[0032] This application also provides the application of the above-described biomarker combination and / or the above-described reagents in the preparation of a kit specifically designed to assess the severity of viremia in patients with chikungunya virus. Standardized diagnostic or assessment kits can be developed using this biomarker combination or detection reagents targeting it.

[0033] Furthermore, the test samples for this kit can include one or more of plasma, serum, and whole blood. Plasma, serum, and whole blood samples are readily available from patients and conform to routine clinical testing procedures. For example, when using plasma as the test sample, it can be obtained directly from the patient's anticoagulated whole blood by centrifugation, and then RNA or protein can be extracted using the components in the kit for subsequent analysis.

[0034] This application also provides a detection product, which includes the above-described biomarker combination and / or the above-described reagents. This detection product is a physical embodiment of biomarker information or a detection tool.

[0035] Furthermore, the detection product can include a chip, such as a gene chip or a protein chip. The chip can be pre-spread with oligonucleotide probes specifically targeting CXCL10 (plasma cytokine), TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1 (plasma mRNA), or capture antibodies targeting these seven proteins. When a processed patient sample (such as labeled cDNA or protein) is hybridized or incubated with the chip, the expression profiles of these six biomarkers can be simultaneously obtained by scanning the signal intensity, thus achieving high-throughput, rapid screening.

[0036] This invention also provides a method for assessing the severity of viremia in patients with chikungunya virus. The method includes the following steps: S1. Detect the expression levels of seven biomarkers in biological samples from patients: CXCL10 (plasma cytokine), TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1 (plasma mRNA). S2. Assess the severity of viremia based on the detected expression levels. Higher expression levels generally indicate more active viral replication and more severe viremia.

[0037] In one specific implementation, the method for detecting the expression level in S1 includes the following steps: S11. Extract total RNA from biological samples; S12. Obtain cDNA from the RNA via reverse transcriptase reaction; S13. Detect the mRNA expression levels of TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1 in this cDNA.

[0038] For example, real-time quantitative polymerase chain reaction (qPCR) can be used for detection: amplification is performed on a quantitative PCR instrument using specific primer pairs and fluorescent probes designed for each biomarker. The relative or absolute expression level of each biomarker is calculated by comparing the cycle threshold of the sample with a standard curve or internal reference gene. Detection can be performed on, for example, an ABI 7500 real-time PCR system, with reaction conditions set as follows: 95°C pre-denaturation for 30 seconds; followed by 40 cycles, each cycle consisting of 95°C denaturation for 5 seconds and 60°C annealing / extension for 34 seconds.

[0039] When assessing severity, the expression level of each biomarker can be compared to a threshold or reference range established in advance through clinical trials. For example, a comprehensive scoring model can be developed that standardizes and weights the expression levels of six biomarkers to obtain a viremia severity index. This index can then be divided into different levels, such as mild, moderate, and severe, to achieve graded assessment.

[0040] The following specific examples provide further details.

[0041] Example 1: Discovery and Screening of mRNA Biomarkers This embodiment included 5 patients infected with Chikungunya virus (including 2 patients with viremia and 3 patients in the recovery period). Informed consent was obtained for all samples and the samples were approved by the ethics committee.

[0042] Five mL of peripheral venous blood (EDTA anticoagulant tube) was collected and centrifuged at 3000 rpm for 10 min at 4°C. The supernatant plasma was separated and stored at -80°C for later use. Total RNA (including host and viral RNA) was extracted from the plasma and used to construct a next-generation sequencing library using the Illumina TruSeq StrandedTotal RNA Library Prep Kit: rRNA was removed, the RNA was randomly fragmented, reverse transcribed into cDNA, ligated with sequencing adapters, and amplified by PCR. The library concentration was quantified using Qubit 4.0, and the fragment size (200-300 bp) was validated using an Agilent 2100. The qualified library was subjected to PE150 paired-end sequencing on the Illumina NovaSeq 6000 platform. Bioinformatics analysis: Trimmomatic filtering of low-quality reads, HISAT2 alignment to the human reference genome (GRCh38), and Bowtie2 alignment to the chikungunya virus genome; featureCounts quantification of gene expression levels, differential analysis using the R package DESeq2, and screening for genes with high expression (|log2FC|>2) in the acute infection group relative to the convalescent group, including TAP1, ISG15, CMPK2, GBP4, OTOF, and SIGLEC1. Further analysis revealed a strong positive correlation between the serum expression levels (FPKM values) of these six genes and viral gene content (FPKM values) (e.g., ...). Figure 1 (As shown). The six genes selected were then used as candidate genes.

[0043] Example 2: qPCR Validation This study also verified the expression of these host genes in serum using real-time quantitative polymerase chain reaction (qPCR). The specific steps were as follows: Table 1. Primer sequence listing

[0044] Total RNA was extracted from plasma samples using TRIzol™ LS Reagent (Invitrogen, #10296010CN), and cDNA was synthesized according to the manufacturer's instructions (Vazyme, #R232). Real-time quantitative PCR was performed using SYBR Green (Vazyme, #Q412) in a 20 μL reaction volume. Cycling conditions included an initial denaturation step of 95°C for 5 min, 45 cycles of 95°C for 10 s and 60°C for 30 s, followed by melting curve analysis. The results also confirmed a high positive correlation between the expression of these host genes and viral gene content (FPKM value) (e.g., ...). Figure 2 (As shown).

[0045] Subsequently, this study examined the messenger RNA levels of these genes in the serum of healthy volunteers and found that the expression of these six genes was almost undetectable in the serum of healthy volunteers compared to infected patients. This further confirms the reliability of these genes as markers of the severity of chikungunya viremia (e.g., Figure 3 (As shown).

[0046] Example 3 Discovery and Screening of Plasma Cytokine Markers Plasma levels of 120 cytokines were measured using a Human Cytokine Array QAH-CAA-2000 (RayBiotech). EDTA-anticoagulated plasma extracted from acute infection and normal groups (n = 3 per group) was centrifuged, aliquoted, and stored at -80°C. After thawing and clarification, the samples were diluted twice with reagent diluent. The array was first blocked, then the diluted plasma was loaded, and incubated overnight at 4°C. After washing, the detection antibody mixture and Cy3-streptavidin were applied sequentially. Fluorescence was scanned (InnoScan 300, 532 nm), and analyte concentrations were calculated from the manufacturer's software based on a standard curve (R² ≥ 0.95; generated using a series of diluted calibrators), while cytokine concentrations in the sample plasma were calculated based on their fluorescence signals. Results are as follows: Figure 4 As shown. Its cutoff criteria are |log2FC|>1 and p<0.05. Figure 4 Medium red dots represent significantly upregulated immune factors in infected patients; gray dots represent immune factors with no significant changes. From Figure 4 It can be seen that the levels of CXCL10 cytokine in the plasma of infected patients are significantly elevated.

[0047] Differential expression analysis of transcripts of 120 immune factor-related genes from all PBMC cell scRNA-seq data, such as Figure 5 and Figure 6As shown (infected group vs. normal group, |log2FC|>1, p<0.05). Red dots represent significantly upregulated immune factors in infected patients; blue dots represent significantly downregulated immune factors; gray dots represent immune factors with no significant change. Figure 5 It can be seen that the expression level of CXCL10 cytokine in PBMC cells of infected patients is significantly increased at the transcriptional level. Figure 6 The data shows that the increase in CXCL0 is the most significant.

[0048] The results were validated using the CXCL10 ELISA kit (Cat. No. 1117452). The results are as follows: Figure 7 As shown, the expression level of CXCL10 in the plasma of infected patients was indeed significantly increased, further validating the reliability of plasma CXCL10 cytokine as a marker of viremia in CHIKV patients.

[0049] In summary, Examples 1-3 explored the characteristics of Chikungunya virus and identified viremia-related biomarkers through viral genetic analysis and plasma messenger RNA profiling: viral sequencing showed that viremia occurred within 3 days after the onset of illness, and 6 host genes (TAP1, ISG15, CMPK2, GBP4, OTOF, SIGLEC1) and 1 cytokine (CXCL10) in serum were verified as reliable auxiliary biomarkers of viremia severity.

[0050] The above description is merely an exemplary embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the technical concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A combination of biomarkers for assessing the severity of viremia in patients with Chikungunya virus, characterized in that, Including one or more of the following biomarkers: TAP1, ISG15, CMPK2, GBP4, OTOF, SIGLEC1.

2. The biomarker combination as described in claim 1, characterized in that, The biomarker also includes: CXCL10.

3. A reagent for detecting the biomarker of claim 2, characterized in that, The reagent comprises a biomolecule that specifically hybridizes with a combination of the biomarkers of claim 2 or their expression products.

4. The reagent as described in claim 3, characterized in that, The biomolecules include one or more of primers, probes, and antibodies.

5. The reagent as described in claim 3, characterized in that, The reagents include those for preparing high-throughput sequencing libraries from the RNA of the biomarker combination as described in claim 2.

6. The use of the biomarker combination of claim 1 or 2 and / or the reagent of any one of claims 3 to 5 in the preparation of the kit, characterized in that, The kit is used to assess the severity of viremia in patients with Chikungunya virus.

7. A testing product, characterized in that, Includes the combination of biomarkers as described in claim 1 or 2 and / or the reagents as described in any one of claims 3 to 5.

8. The test product as described in claim 7, characterized in that, The testing products include one or more types of chips.

9. A method for assessing the severity of viremia in patients with Chikungunya virus, characterized in that, The method includes the following steps: detecting the expression level of the combination of biomarkers as described in claim 1 or 2 in a biological sample from a patient; and assessing the severity of viremia based on the expression level.

10. The method as described in claim 9, characterized in that, The method for detecting the expression level includes: extracting RNA from the biological sample; reverse transcribing the RNA to obtain cDNA; and detecting the mRNA expression level of the biomarker combination of claim 1 in the cDNA.

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