Molecular detection of NSP-5 and its relation with mi-RNA from patient infected with Rotavirus in Baghdad City

Molecular detection of NSP-5 and its relation with mi-RNA from patient infected with Rotavirus in Baghdad City | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular detection of NSP-5 and its relation with mi-RNA from patient infected with Rotavirus in Baghdad City Mohammed T. Jaber, Alyaa M. Zyara, Zahraa M. AL-Jumaa, Atheer A. AL-Doori, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8058136/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Vaccination is still the only effective way to prevent acute diarrhea in children under five, which is primarily caused by rotavirus. It is essential to look into virus-host interactions and find new biomarkers for early diagnosis in the absence of targeted treatments. The relationship between rotavirus infection and microRNA-7 (miR-7) expression is investigated in this work. Methods: 130 stool samples from children with acute gastroenteritis under the age of five were gathered. A commercial rapid antigen test was first used to screen for rotavirus infection, yielding 27 positive and 103 negative samples. All samples had their total RNA extracted, and RT-qPCR was used to measure the expression levels of miR-7, using miR-16 as an endogenous control. Receiver Operating Characteristic (ROC) curve analysis was used to evaluate miR-7's diagnostic performance. Additionally, the rotavirus NSP5 and NSP3 genes were found in a subset of samples using traditional RT-PCR. Findings: In stools from the rapid-test-positive group (n = 27) compared to the negative disease-control group (n = 103), MiR-7 expression was significantly upregulated (p < 0.0001). MiR-7 has a high diagnostic accuracy for differentiating these groups, according to ROC curve analysis, with an area under the curve (AUC) of 0.965, 85% sensitivity, and 99% specificity at an ideal cut-off of 5.83-fold change. Our particular RT-PCR assays detected the NSP5 gene in only 4 samples (3.1%) and the NSP3 gene in 1 sample (0.76%), despite the rapid test showing a positivity rate of 20.8%. This suggests a significant discrepancy that is probably caused by high primer specificity for specific rotavirus strains that are not prevalent in our cohort. Conclusion: Children with gastroenteritis who test positive for rotavirus using a rapid antigen test have significantly higher levels of fecal miR-7. In this situation, it exhibits high diagnostic accuracy. It is a promising non-invasive biomarker due to its stability in stool. Future research employing more comprehensive molecular confirmation techniques (such as RT-qPCR targeting conserved genes) is necessary to conclusively establish miR-7's specificity for rotavirus infection, as the low confirmation rate by our particular RT-PCR assays indicates the association is with the rapid test status. miR-7 Rotavirus Stool samples Biomarker Gene expression NSP5 Diagnosis Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Worldwide, rotavirus is the primary cause of severe acute viral gastroenteritis in infants and young children, which greatly increases morbidity and mortality from dehydration [ 1 ]. When the virus infects mature enterocytes in the small intestine, it binds to host cell receptors like integrins and sialic acid to start its replication cycle, which is then followed by endocytosis [ 2 ]. The gut's ability to absorb nutrients is hampered by this infection. Additionally, by releasing the enterotoxin NSP4, which promotes fluid and electrolyte secretion, the virus makes diarrhea worse [ 1 , 3 ]. Acute watery diarrhea, the classic sign of rotavirus infection, is caused by both increased secretion and impaired absorption. Eleven double-stranded RNA segments make up the rotavirus genome. The crucial non-structural proteins NSP2 and NSP5 form viroplasms, which are cytoplasmic inclusions where viral replication is coordinated [ 4 ]. As a scaffold protein, NSP5 arranges viral components for particle assembly and genome replication; the formation of viroplasm depends on its phosphorylation state [ 5 ]. By dislodging the cellular poly (A)-binding protein (PABP) from the translation initiation factor eIF4G, another important non-structural protein, NSP3, promotes the selective translation of viral mRNAs and successfully takes over the host's cellular machinery to enable viral protein synthesis [ 6 , 7 ]. MicroRNAs (miRNAs) have become important modulators of gene expression in the interaction between the virus and its host. The 3' untranslated regions of target mRNAs are usually bound by these tiny, non-coding RNAs, which causes translational repression or mRNA degradation [ 8 ]. Numerous biological processes, such as immune responses, cell division, and proliferation, are influenced by miRNAs [ 9 ]. Notably, miRNAs are remarkably stable in bodily fluids such as feces, as they are protected within exosomes or protein complexes, making them excellent candidates for non-invasive biomarkers for various diseases [ 10 ]. MiR-7 is one of these miRNAs that has been linked to controlling inflammatory and immune responses [ 9 ]. Crucially, an increasing amount of data emphasizes its direct contribution to antiviral defense. By specifically targeting the NSP5 gene, a vital part of the viral replication machinery, miR-7 prevents rotavirus replication, according to a recent study [ 11 ]. This implies that the host's innate immune response to rotavirus infection may include the upregulation of miR-7. MiR-7 is a promising non-invasive diagnostic biomarker for rotavirus infection because of its specific antiviral activity and remarkable stability in feces. A quick stool-based diagnostic test might be developed if rotavirus-associated gastroenteritis patients have markedly elevated fecal miR-7 levels. Therefore, the purpose of this study is to examine the detection of viral NSP genes and the relationship between miR-7 expression and rotavirus infection as assessed by a clinical rapid test. Aims of the Study The study's objectives are as follows: (1) Diagnostic Biomarker Validation : By comparing the expression levels of miR-7 in acute-test-positive and negative gastroenteritis patients, this study aims to assess the potential of miR-7 as a non-invasive fecal biomarker for rotavirus infection. (2) Clinical Correlation : To assess the expression levels of miR-7 in stool samples from children who have gastroenteritis and who test positive or negative for the rotavirus rapid antigen test. (3) Viral Gene Detection : To identify and document if rotavirus NSP3 and NSP5 genes are present in a subset of stool samples that were gathered. MATERIALS AND METHODS Patient Samples and Ethical Approval Children under five who presented with acute gastroenteritis symptoms (watery diarrhea, with or without vomiting and fever) at participating clinics in Baghdad were asked to provide 130 fecal samples. All legal guardians gave their informed consent, and the study was approved by the Higher Institute of Forensic Sciences' Institutional Review Board at Al-Nahrain University. The samples were kept at -80°C until analysis and were delivered to the lab in chilled containers at 4°C. RNA Extraction from Stool Following the manufacturer's instructions, TRIzol™ Reagent (Invitrogen, USA) was used to extract total RNA, including miRNA, from about 100 mg of stool. In short, samples were phase separated using chloroform after being homogenized in TRIzol. Isopropanol was used to precipitate the RNA, followed by 75% ethanol washing and RNase-free water dissolution. RNA concentration and purity were measured using the Quantus™ Fluorometer (Promega, USA) equipped with the Quantifluor™ RNA System. Acceptable samples had a 260/280 ratio of ≥ 1.8. miRNA Reverse Transcription and Quantitative PCR (RT-qPCR) Using the OT-1 Reagent Kit, a poly-A tailing protocol, and the MMLV-RT enzyme, cDNA was created from total RNA for miRNA analysis. Synthol (Russia) Sybr Green I Master Mix (2.5X) was used for the ensuing qPCR on a SimpliAmpTM Thermal Cycler (Applied Biosystems, USA). The amplification program consisted of 10 minutes at 95°C, 40 cycles of 20 seconds at 95°C, 20 seconds at 56°C, and 20 seconds at 72°C. The endogenous control for normalization was miR-16. The primer sequences listed below were employed: miR-7-5p: 5'-UGGAAGACUAGUGAUUUUGUUGU-3' miR-16: 5'-UAGCAGCACGUAAAUAUUGGCG-3' Expression levels of miR-7-5p were calculated using the 2^(-ΔΔCt) method [ 12 ]. Rotavirus Detection by Rapid Test and RT-PCR Initially, a commercial rapid antigen immunochromatographic test was used to screen for rotavirus infection. Conventional RT-PCR was used for molecular detection. MMLV-RT and random hexamers were used to create cDNA from extracted RNA. The NSP5 gene was then the target of PCR, which produced a 550 bp amplicon using particular primers (Forward: 5'-ATGAGCACAATAGTTAAAAGCTAAC-3'; Reverse: 5'-AATCTGTAACACATACTCCAC-3'). A subset of reactions also targeted the NSP3 gene (1063 bp). A 100-bp DNA ladder and a 2% agarose gel stained with ethidium bromide were used to view the PCR results. Statistical Analysis The mean and standard deviation of miR-7 gene expression levels were calculated using R (version 4.3.2). A comparison between rapid-test-positive and negative groups was performed using an independent-samples t-test. A receiver operating characteristic (ROC) curve was generated to evaluate the diagnostic potential of miR-7 for distinguishing these two groups. The area under the curve (AUC) and Youden's criterion were used to determine the optimal cut-off point, sensitivity, and specificity. A p-value of less than 0.05 was considered statistically significant. RESULTS miR-7 Expression is Significantly Upregulated in Rapid-Test-Positive Stools The expression level of miR-7 was significantly higher in rapid-test-positive stool samples (n=27) compared to negative disease controls (n=103). The T-test analysis confirmed a statistically significant difference between the groups (P < 0.0001) (Figure 1). Figure 1: Difference in miR-7 gene expression between rapid-test-positive and negative stool samples. Data are presented as a box-and-whisker plot. The y-axis represents miR-7 Fold Change (2^–ΔΔCt). The difference was statistically significant (unpaired t-test, p < 0.0001). Figure 1: Difference in miR-7 gene expression between rotavirus-infected and uninfected stool samples. Data are presented as a box-and-whisker plot. The difference was statistically significant (unpaired t-test, p < 0.0001). Diagnostic Performance of Fecal miR-7 The ROC curve analysis for miR-7's ability to distinguish rapid-test-positive from negative samples yielded an AUC of 0.965, indicating excellent diagnostic accuracy for this specific comparison (Figure 2). The optimal threshold for miR-7 expression was established at 5.83-fold change, yielding a sensitivity of 85% and a specificity of 99%. Figure 2: ROC Curve analysis of miR-7 expression for distinguishing rapid-test-positive from negative samples. The Area Under the Curve (AUC) is 0.965. Detection of Rotavirus Genes NSP5 and NSP3 Among the 130 samples, 27 (20.8%) were positive by a commercial rapid antigen test. However, using our in-house RT-PCR, the NSP5 gene was detected in only 4 samples (3.1%), and the NSP3 gene was detected in 1 sample (0.76%) (Figure 3). All PCR-positive samples were also positive by the rapid test. Figure 3: Detection of viral genes NSP3 and NSP5 in stool samples by conventional RT-PCR Figure 4 shows a representative agarose gel of RT-PCR products. A distinct amplicon at 550 bp confirms the presence of the NSP5 gene (Lanes 3, 4, 5), and a band at 1063 bp confirms the NSP3 gene (Lane 1). Lane M shows the DNA molecular weight marker. Figure 4: Agarose gel electrophoresis of RT-PCR products for Rotavirus NSP3 and NSP5 genes. Lane M: DNA molecular weight marker. Lane 1: Positive sample for the NSP3 gene (1063 bp). Lanes 3, 4, 5: Positive samples for the NSP5 gene (550 bp). DISCUSSION In children with gastroenteritis who tested positive for rotavirus using a rapid antigen test, this study shows a significant upregulation of fecal miR-7, indicating its potential as a diagnostic biomarker in this situation. Promising results include the significant difference in miR-7 expression between the two patient groups and its high diagnostic accuracy (AUC = 0.965). The host's natural antiviral defense may be responsible for the observed increase in fecal miR-7. According to earlier molecular research, rotavirus infection modifies the production of miRNAs in host cells, with miR-7 being one of the microRNAs that is markedly induced [ 11 , 13 ]. Importantly, the mechanism underlying this response is well-established: miR-7 directly targets the mRNA of the viral NSP5 gene, a crucial protein required for the development of viroplasms and viral replication [ 11 ]. Consequently, a targeted cellular attempt to inhibit viral replication may be indicated by the elevated levels of miR-7 in stool. Its suitability as a non-invasive biomarker is further enhanced by the stability of miR-7 in feces, which is shielded from degradation within exosomes or protein complexes [ 10 ]. The significant difference between our RT-PCR detection of NSP5/NSP3 and the commercial rapid antigen test was a key finding in our investigation. This is a significant drawback that may be caused by primer mismatch and the higher specificity of PCR, among other things. Certain rotavirus strains that were not the most common strains circulating in our patient cohort during the study period may be highly specific for the NSP5 and NSP3 primers used in this investigation. Because of the high genetic diversity of the virus, this is a frequent problem in rotavirus molecular epidemiology. Therefore, the rapid test defines the "rotavirus-positive" group for the miR-7 analysis, and because of the low PCR confirmation rate, we are unable to completely rule out the possibility that some rapid-test-positive samples were false positives or contained strains that our primers were unable to detect. Future research using more comprehensive molecular screening techniques (such as RT-qPCR for the conserved VP6 gene) is therefore required to confirm the association with conclusively confirmed rotavirus infection, as the association we report is specific to miR-7 and rapid-test status. The absence of a robust control group is another drawback. The fact that children in our "negative" group had gastroenteritis from other sources supports the idea that miR-7 is linked to rotavirus rather than other diarrheal etiologies. We are unable to create a reliable baseline or rule out non-specific upregulation brought on by general intestinal inflammation, though, in the absence of healthy controls. There are other restrictions on our study. A significant drawback of using traditional, qualitative PCR for viral detection is that it is less sensitive and specific than the suggested RT-qPCR, and it does not allow for the correlation of miR-7 levels with a quantitative viral load. Quantitative RT-PCR (RT-qPCR) should be used in future research to investigate this relationship. Finally, the current reliance on RT-qPCR for miR-7 quantification may be less practical for point-of-care settings compared to rapid antigen tests. Future efforts should focus on developing simpler, faster detection platforms, such as lateral flow assays, to translate this biomarker into clinical utility. CONCLUSION Rotavirus-Positive Samples Show Upregulated miR-7 : Compared to rapid-test-negative controls with gastroenteritis, stool samples from children who tested positive for rotavirus by a rapid antigen test showed significantly higher miR-7 expression (p < 0.0001). Promising Diagnostic Biomarker : miR-7 showed remarkable diagnostic accuracy (AUC = 0.965) with 85% sensitivity and 99% specificity for differentiating rapid-test-positive from negative samples, confirming its potential as a non-invasive screening method. Methodological Difference in Viral Detection : A notable difference between our in-house RT-PCR and the commercial rapid test was found, indicating that the rapid test status is related to the association. The association between miR-7 and a rotavirus infection that has been definitively confirmed needs to be validated using more reliable molecular techniques because of the low PCR confirmation rate, which may be caused by primer mismatch with circulating strains. Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Conflict of interest All authors declare that they have no conflicts of interest. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Review Board of the Higher Institute of Forensic Sciences, Al-Nahrain University. Consent to participate Informed consent was obtained from all legal guardians of individual participants included in the study. Consent for publication All authors have read and approved the final manuscript for publication. Data availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. Code availability Not applicable. Authors' contributions Mohammed T. Jaber: Conceptualization, Methodology, Writing - Review & Editing, Supervision. Alyaa M. Zyara: Formal analysis, Investigation, Data Curation, Writing - Original Draft. Zahraa M. AL-Jumaa: Methodology, Validation, Resources. Atheer A. AL-Doori: Investigation, Resources. Mariam H. Abdulkareem: Investigation, Resources. Acknowledgements The authors would like to thank the staff and technicians at the Department of Forensic Biology and the Department of Microbiology for their technical support and assistance. References Crawford, S. E., Ramani, S., Tate, J. E., Parashar, U. D., Svensson, L., Hagbom, M., ... & Estes, M. K. (2017). Rotavirus infection. Nature Reviews Disease Primers, 3(1), 17083. Rodríguez, D. F. H. (2020). Study of the human humoral immune response against rotavirus. Ball, J. M., Tian, P., Zeng, C. Q., Morris, A. P., & Estes, M. K. (1996). Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science, 272(5258), 101-104. Contin, R., Arnoldi, F., Campagna, M., & Burrone, O. R. (2010). Rotavirus NSP5 orchestrates recruitment of viroplasmic proteins. Journal of General Virology, 91(7), 1782-1793. Silvestri, L. S., Taraporewala, Z. F., & Patton, J. T. (2004). Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms. Journal of Virology, 78(14), 7763-7774. Piron, M., Vende, P., Cohen, J., & Poncet, D. (1998). Rotavirus RNA‐binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F. The EMBO Journal, 17(19), 5811-5821. Poncet, D., Aponte, C., & Cohen, J. (1993). Rotavirus protein NSP3 (NS34) is bound to the 3' end consensus sequence of viral mRNAs in infected cells. Journal of Virology, 67(6), 3159-3165. Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2), 281-297. Chen, C., Guo, M., Zhao, X., Zhao, J., Chen, L., He, Z., ... & Zha, Y. (2023). MicroRNA-7: a new intervention target for inflammation and related diseases. Biomolecules, 13(8), 1185. Rashid, H., Hossain, B., Siddiqua, T., Kabir, M., Noor, Z., Ahmed, M., & Haque, R. (2020). Fecal microRNAs as potential biomarkers for screening and diagnosis of intestinal diseases. Frontiers in Molecular Biosciences, 7, 181. Zhou, Y., Chen, L., Du, J., Hu, X., Xie, Y., Wu, J. & Li, H. (2020). MicroRNA-7 inhibits rotavirus replication by targeting viral NSP5 in vivo and in vitro. Viruses, 12(2), 209. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001;25(4):402-8. Al-dabbagh, Y. A., Rasheed, B. Y. and AL-Jumaa, Z. M. (2021). Molecular diagnosis of Mycoplasma gallisepticum in turkey in Mosul city. Veterinary Practitioner, 22(1), 1-4. Al-Jumaa, Z. M., Jaber, M. T., & Al-Doori, A. A. (2024). Molecular detection of Chlamydophila felis from conjunctiva of cats infected with conjunctivitis and upper respiratory disease. Open Veterinary Journal, 14(12), 3289. https://doi.org/10.5455/OVJ.2024.v14.i12.13 Papa, G., Borodavka, A., & Desselberger, U. (2021). Viroplasms: assembly and functions of rotavirus replication factories. Viruses, 13(7), 1349. Zyara, A. M., Aldoori, A. A., Samawi, F. T., Kadhim, S. I., & Ali, Z. A. (2023). A relationship study of coronavirus (COVID-19) infection, blood groups, and some related factors in Iraqi patients. Baghdad Science Journal, 20(4), 32. https://doi.org/10.21123/bsj.2023.8871 Additional Declarations No competing interests reported. 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16:01:55","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46045,"visible":true,"origin":"","legend":"","description":"","filename":"6d5df06327c941a98ab24058939cd2331structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8058136/v1/ca94fde30e5d669609a25d51.xml"},{"id":96399750,"identity":"709eaf67-6e82-493e-9cae-ddf685b6db0d","added_by":"auto","created_at":"2025-11-20 16:01:55","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":54277,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8058136/v1/3aa45fc6bb4852e4d623d4f3.html"},{"id":96399730,"identity":"7f4a0f2a-3c71-4dab-83ff-f6b388c62667","added_by":"auto","created_at":"2025-11-20 16:01:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifference in miR-7 gene expression between rotavirus-infected and uninfected stool samples.\u003c/strong\u003e Data are presented as a box-and-whisker plot. The difference was statistically significant (unpaired t-test, p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8058136/v1/30faa70dcba714ad3d4880bf.png"},{"id":96399736,"identity":"157e8371-883d-4eaa-84fd-d148acd1672f","added_by":"auto","created_at":"2025-11-20 16:01:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34671,"visible":true,"origin":"","legend":"\u003cp\u003eROC Curve analysis of miR-7 expression for distinguishing rapid-test-positive from negative samples. The Area Under the Curve (AUC) is 0.965.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8058136/v1/ab49ea467cfc212956ae2788.png"},{"id":96399733,"identity":"996659b8-0096-48b1-9266-c82bed1bdc38","added_by":"auto","created_at":"2025-11-20 16:01:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of viral genes NSP3 and NSP5 in stool samples by conventional RT-PCR\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8058136/v1/9ed48fbdf8d9a93fe7edf532.png"},{"id":96454345,"identity":"b80fdfb7-f461-4ed1-ba8f-214ed2ad8976","added_by":"auto","created_at":"2025-11-21 10:02:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":220318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAgarose gel electrophoresis of RT-PCR products for Rotavirus NSP3 and NSP5 genes.\u003c/strong\u003e Lane M: DNA molecular weight marker. Lane 1: Positive sample for the NSP3 gene (1063 bp). Lanes 3, 4, 5: Positive samples for the NSP5 gene (550 bp).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8058136/v1/c832977800913984ad9fbbed.png"},{"id":97668032,"identity":"bc2b608f-036c-49f7-98e3-14ad9592abb9","added_by":"auto","created_at":"2025-12-08 09:24:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1013351,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8058136/v1/b0773c8c-1c75-4ee0-836e-bdc610fd4757.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular detection of NSP-5 and its relation with mi-RNA from patient infected with Rotavirus in Baghdad City","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eWorldwide, rotavirus is the primary cause of severe acute viral gastroenteritis in infants and young children, which greatly increases morbidity and mortality from dehydration [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. When the virus infects mature enterocytes in the small intestine, it binds to host cell receptors like integrins and sialic acid to start its replication cycle, which is then followed by endocytosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The gut's ability to absorb nutrients is hampered by this infection. Additionally, by releasing the enterotoxin NSP4, which promotes fluid and electrolyte secretion, the virus makes diarrhea worse [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Acute watery diarrhea, the classic sign of rotavirus infection, is caused by both increased secretion and impaired absorption.\u003c/p\u003e\u003cp\u003eEleven double-stranded RNA segments make up the rotavirus genome. The crucial non-structural proteins NSP2 and NSP5 form viroplasms, which are cytoplasmic inclusions where viral replication is coordinated [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As a scaffold protein, NSP5 arranges viral components for particle assembly and genome replication; the formation of viroplasm depends on its phosphorylation state [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. By dislodging the cellular poly (A)-binding protein (PABP) from the translation initiation factor eIF4G, another important non-structural protein, NSP3, promotes the selective translation of viral mRNAs and successfully takes over the host's cellular machinery to enable viral protein synthesis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMicroRNAs (miRNAs) have become important modulators of gene expression in the interaction between the virus and its host. The 3' untranslated regions of target mRNAs are usually bound by these tiny, non-coding RNAs, which causes translational repression or mRNA degradation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Numerous biological processes, such as immune responses, cell division, and proliferation, are influenced by miRNAs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, miRNAs are remarkably stable in bodily fluids such as feces, as they are protected within exosomes or protein complexes, making them excellent candidates for non-invasive biomarkers for various diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMiR-7 is one of these miRNAs that has been linked to controlling inflammatory and immune responses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Crucially, an increasing amount of data emphasizes its direct contribution to antiviral defense. By specifically targeting the NSP5 gene, a vital part of the viral replication machinery, miR-7 prevents rotavirus replication, according to a recent study [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This implies that the host's innate immune response to rotavirus infection may include the upregulation of miR-7.\u003c/p\u003e\u003cp\u003eMiR-7 is a promising non-invasive diagnostic biomarker for rotavirus infection because of its specific antiviral activity and remarkable stability in feces. A quick stool-based diagnostic test might be developed if rotavirus-associated gastroenteritis patients have markedly elevated fecal miR-7 levels. Therefore, the purpose of this study is to examine the detection of viral NSP genes and the relationship between miR-7 expression and rotavirus infection as assessed by a clinical rapid test.\u003c/p\u003e\n\u003ch3\u003eAims of the Study\u003c/h3\u003e\n\u003cp\u003eThe study's objectives are as follows: \u003cb\u003e(1) Diagnostic Biomarker Validation\u003c/b\u003e: By comparing the expression levels of miR-7 in acute-test-positive and negative gastroenteritis patients, this study aims to assess the potential of miR-7 as a non-invasive fecal biomarker for rotavirus infection.\u003c/p\u003e\u003cp\u003e\u003cb\u003e(2) Clinical Correlation\u003c/b\u003e: To assess the expression levels of miR-7 in stool samples from children who have gastroenteritis and who test positive or negative for the rotavirus rapid antigen test.\u003c/p\u003e\u003cp\u003e\u003cb\u003e(3) Viral Gene Detection\u003c/b\u003e: To identify and document if rotavirus NSP3 and NSP5 genes are present in a subset of stool samples that were gathered.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003ePatient Samples and Ethical Approval\u003c/h2\u003e\u003cp\u003eChildren under five who presented with acute gastroenteritis symptoms (watery diarrhea, with or without vomiting and fever) at participating clinics in Baghdad were asked to provide 130 fecal samples. All legal guardians gave their informed consent, and the study was approved by the Higher Institute of Forensic Sciences' Institutional Review Board at Al-Nahrain University. The samples were kept at -80\u0026deg;C until analysis and were delivered to the lab in chilled containers at 4\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA Extraction from Stool\u003c/h3\u003e\n\u003cp\u003eFollowing the manufacturer's instructions, TRIzol\u0026trade; Reagent (Invitrogen, USA) was used to extract total RNA, including miRNA, from about 100 mg of stool. In short, samples were phase separated using chloroform after being homogenized in TRIzol. Isopropanol was used to precipitate the RNA, followed by 75% ethanol washing and RNase-free water dissolution. RNA concentration and purity were measured using the Quantus\u0026trade; Fluorometer (Promega, USA) equipped with the Quantifluor\u0026trade; RNA System. Acceptable samples had a 260/280 ratio of \u0026ge;\u0026thinsp;1.8.\u003c/p\u003e\n\u003ch3\u003emiRNA Reverse Transcription and Quantitative PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eUsing the OT-1 Reagent Kit, a poly-A tailing protocol, and the MMLV-RT enzyme, cDNA was created from total RNA for miRNA analysis. Synthol (Russia) Sybr Green I Master Mix (2.5X) was used for the ensuing qPCR on a SimpliAmpTM Thermal Cycler (Applied Biosystems, USA). The amplification program consisted of 10 minutes at 95\u0026deg;C, 40 cycles of 20 seconds at 95\u0026deg;C, 20 seconds at 56\u0026deg;C, and 20 seconds at 72\u0026deg;C. The endogenous control for normalization was miR-16. The primer sequences listed below were employed:\u003c/p\u003e\n\u003ch3\u003emiR-7-5p: 5'-UGGAAGACUAGUGAUUUUGUUGU-3'\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003emiR-16: 5'-UAGCAGCACGUAAAUAUUGGCG-3'\u003c/h2\u003e\u003cp\u003eExpression levels of miR-7-5p were calculated using the 2^(-ΔΔCt) method [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRotavirus Detection by Rapid Test and RT-PCR\u003c/h3\u003e\n\u003cp\u003eInitially, a commercial rapid antigen immunochromatographic test was used to screen for rotavirus infection. Conventional RT-PCR was used for molecular detection. MMLV-RT and random hexamers were used to create cDNA from extracted RNA. The NSP5 gene was then the target of PCR, which produced a 550 bp amplicon using particular primers (Forward: 5'-ATGAGCACAATAGTTAAAAGCTAAC-3'; Reverse: 5'-AATCTGTAACACATACTCCAC-3'). A subset of reactions also targeted the NSP3 gene (1063 bp). A 100-bp DNA ladder and a 2% agarose gel stained with ethidium bromide were used to view the PCR results.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe mean and standard deviation of miR-7 gene expression levels were calculated using R (version 4.3.2). A comparison between rapid-test-positive and negative groups was performed using an independent-samples t-test. A receiver operating characteristic (ROC) curve was generated to evaluate the diagnostic potential of miR-7 for distinguishing these two groups. The area under the curve (AUC) and Youden's criterion were used to determine the optimal cut-off point, sensitivity, and specificity. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003emiR-7 Expression is Significantly Upregulated in Rapid-Test-Positive Stools\u003c/p\u003e\n\u003cp\u003eThe expression level of miR-7 was significantly higher in rapid-test-positive stool samples (n=27) compared to negative disease controls (n=103). The T-test analysis confirmed a statistically significant difference between the groups (P \u0026lt; 0.0001) (Figure 1).\u003c/p\u003e\n\u003cp\u003eFigure 1:\u0026nbsp;Difference in miR-7 gene expression between rapid-test-positive and negative stool samples. Data are presented as a box-and-whisker plot. The y-axis represents miR-7 Fold Change (2^\u0026ndash;\u0026Delta;\u0026Delta;Ct). The difference was statistically significant (unpaired t-test, p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003eFigure 1: Difference in miR-7 gene expression between rotavirus-infected and uninfected stool samples. Data are presented as a box-and-whisker plot. The difference was statistically significant (unpaired t-test, p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003eDiagnostic Performance of Fecal miR-7\u003cbr\u003e\u0026nbsp;The ROC curve analysis for miR-7\u0026apos;s ability to distinguish rapid-test-positive from negative samples yielded an AUC of 0.965, indicating excellent diagnostic accuracy for this specific comparison (Figure 2). The optimal threshold for miR-7 expression was established at 5.83-fold change, yielding a sensitivity of 85% and a specificity of 99%.\u003c/p\u003e\n\u003cp\u003eFigure 2:\u0026nbsp;ROC Curve analysis of miR-7 expression for distinguishing rapid-test-positive from negative samples. The Area Under the Curve (AUC) is 0.965.\u003c/p\u003e\n\u003cp\u003eDetection of Rotavirus Genes NSP5 and NSP3\u003cbr\u003e\u0026nbsp;Among the 130 samples, 27 (20.8%) were positive by a commercial rapid antigen test. However, using our in-house RT-PCR, the NSP5 gene was detected in only 4 samples (3.1%), and the NSP3 gene was detected in 1 sample (0.76%) (Figure 3). All PCR-positive samples were also positive by the rapid test.\u003c/p\u003e\n\u003cp\u003eFigure 3: Detection of viral genes NSP3 and NSP5 in stool samples by conventional RT-PCR\u003c/p\u003e\n\u003cp\u003eFigure 4\u0026nbsp;shows a representative agarose gel of RT-PCR products. A distinct amplicon at 550 bp confirms the presence of the NSP5 gene (Lanes 3, 4, 5), and a band at 1063 bp confirms the NSP3 gene (Lane 1). Lane M shows the DNA molecular weight marker.\u003c/p\u003e\n\u003cp\u003eFigure 4: Agarose gel electrophoresis of RT-PCR products for Rotavirus NSP3 and NSP5 genes. Lane M: DNA molecular weight marker. Lane 1: Positive sample for the NSP3 gene (1063 bp). Lanes 3, 4, 5: Positive samples for the NSP5 gene (550 bp).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn children with gastroenteritis who tested positive for rotavirus using a rapid antigen test, this study shows a significant upregulation of fecal miR-7, indicating its potential as a diagnostic biomarker in this situation. Promising results include the significant difference in miR-7 expression between the two patient groups and its high diagnostic accuracy (AUC\u0026thinsp;=\u0026thinsp;0.965).\u003c/p\u003e\u003cp\u003eThe host's natural antiviral defense may be responsible for the observed increase in fecal miR-7. According to earlier molecular research, rotavirus infection modifies the production of miRNAs in host cells, with miR-7 being one of the microRNAs that is markedly induced [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Importantly, the mechanism underlying this response is well-established: miR-7 directly targets the mRNA of the viral NSP5 gene, a crucial protein required for the development of viroplasms and viral replication [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Consequently, a targeted cellular attempt to inhibit viral replication may be indicated by the elevated levels of miR-7 in stool. Its suitability as a non-invasive biomarker is further enhanced by the stability of miR-7 in feces, which is shielded from degradation within exosomes or protein complexes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe significant difference between our RT-PCR detection of NSP5/NSP3 and the commercial rapid antigen test was a key finding in our investigation. This is a significant drawback that may be caused by primer mismatch and the higher specificity of PCR, among other things. Certain rotavirus strains that were not the most common strains circulating in our patient cohort during the study period may be highly specific for the NSP5 and NSP3 primers used in this investigation. Because of the high genetic diversity of the virus, this is a frequent problem in rotavirus molecular epidemiology. Therefore, the rapid test defines the \"rotavirus-positive\" group for the miR-7 analysis, and because of the low PCR confirmation rate, we are unable to completely rule out the possibility that some rapid-test-positive samples were false positives or contained strains that our primers were unable to detect. Future research using more comprehensive molecular screening techniques (such as RT-qPCR for the conserved VP6 gene) is therefore required to confirm the association with conclusively confirmed rotavirus infection, as the association we report is specific to miR-7 and rapid-test status.\u003c/p\u003e\u003cp\u003eThe absence of a robust control group is another drawback. The fact that children in our \"negative\" group had gastroenteritis from other sources supports the idea that miR-7 is linked to rotavirus rather than other diarrheal etiologies. We are unable to create a reliable baseline or rule out non-specific upregulation brought on by general intestinal inflammation, though, in the absence of healthy controls.\u003c/p\u003e\u003cp\u003eThere are other restrictions on our study. A significant drawback of using traditional, qualitative PCR for viral detection is that it is less sensitive and specific than the suggested RT-qPCR, and it does not allow for the correlation of miR-7 levels with a quantitative viral load. Quantitative RT-PCR (RT-qPCR) should be used in future research to investigate this relationship. Finally, the current reliance on RT-qPCR for miR-7 quantification may be less practical for point-of-care settings compared to rapid antigen tests. Future efforts should focus on developing simpler, faster detection platforms, such as lateral flow assays, to translate this biomarker into clinical utility.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003e\u003cb\u003eRotavirus-Positive Samples Show Upregulated miR-7\u003c/b\u003e: Compared to rapid-test-negative controls with gastroenteritis, stool samples from children who tested positive for rotavirus by a rapid antigen test showed significantly higher miR-7 expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePromising Diagnostic Biomarker\u003c/b\u003e: miR-7 showed remarkable diagnostic accuracy (AUC\u0026thinsp;=\u0026thinsp;0.965) with 85% sensitivity and 99% specificity for differentiating rapid-test-positive from negative samples, confirming its potential as a non-invasive screening method.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethodological Difference in Viral Detection\u003c/b\u003e: A notable difference between our in-house RT-PCR and the commercial rapid test was found, indicating that the rapid test status is related to the association. The association between miR-7 and a rotavirus infection that has been definitively confirmed needs to be validated using more reliable molecular techniques because of the low PCR confirmation rate, which may be caused by primer mismatch with circulating strains.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Review Board of the Higher Institute of Forensic Sciences, Al-Nahrain University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Informed consent was obtained from all legal guardians of individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All authors have read and approved the final manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Mohammed T. Jaber: Conceptualization, Methodology, Writing - Review \u0026amp; Editing, Supervision. Alyaa M. Zyara: Formal analysis, Investigation, Data Curation, Writing - Original Draft. Zahraa M. AL-Jumaa: Methodology, Validation, Resources. Atheer A. AL-Doori: Investigation, Resources. Mariam H. Abdulkareem: Investigation, Resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The authors would like to thank the staff and technicians at the Department of Forensic Biology and the Department of Microbiology for their technical support and assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCrawford, S. E., Ramani, S., Tate, J. E., Parashar, U. D., Svensson, L., Hagbom, M., ... \u0026amp; Estes, M. K. (2017). Rotavirus infection. Nature Reviews Disease Primers, 3(1), 17083.\u003c/li\u003e\n \u003cli\u003eRodr\u0026iacute;guez, D. F. H. (2020). Study of the human humoral immune response against rotavirus.\u003c/li\u003e\n \u003cli\u003eBall, J. M., Tian, P., Zeng, C. Q., Morris, A. P., \u0026amp; Estes, M. K. (1996). Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science, 272(5258), 101-104.\u003c/li\u003e\n \u003cli\u003eContin, R., Arnoldi, F., Campagna, M., \u0026amp; Burrone, O. R. (2010). Rotavirus NSP5 orchestrates recruitment of viroplasmic proteins. Journal of General Virology, 91(7), 1782-1793.\u003c/li\u003e\n \u003cli\u003eSilvestri, L. S., Taraporewala, Z. F., \u0026amp; Patton, J. T. (2004). Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms. Journal of Virology, 78(14), 7763-7774.\u003c/li\u003e\n \u003cli\u003ePiron, M., Vende, P., Cohen, J., \u0026amp; Poncet, D. (1998). Rotavirus RNA‐binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F. The EMBO Journal, 17(19), 5811-5821.\u003c/li\u003e\n \u003cli\u003ePoncet, D., Aponte, C., \u0026amp; Cohen, J. (1993). Rotavirus protein NSP3 (NS34) is bound to the 3\u0026apos; end consensus sequence of viral mRNAs in infected cells. Journal of Virology, 67(6), 3159-3165.\u003c/li\u003e\n \u003cli\u003eBartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2), 281-297.\u003c/li\u003e\n \u003cli\u003eChen, C., Guo, M., Zhao, X., Zhao, J., Chen, L., He, Z., ... \u0026amp; Zha, Y. (2023). MicroRNA-7: a new intervention target for inflammation and related diseases. Biomolecules, 13(8), 1185.\u003c/li\u003e\n \u003cli\u003eRashid, H., Hossain, B., Siddiqua, T., Kabir, M., Noor, Z., Ahmed, M., \u0026amp; Haque, R. (2020). Fecal microRNAs as potential biomarkers for screening and diagnosis of intestinal diseases. Frontiers in Molecular Biosciences, 7, 181.\u003c/li\u003e\n \u003cli\u003eZhou, Y., Chen, L., Du, J., Hu, X., Xie, Y., Wu, J. \u0026amp; Li, H. (2020). MicroRNA-7 inhibits rotavirus replication by targeting viral NSP5 in vivo and in vitro. Viruses, 12(2), 209.\u003c/li\u003e\n \u003cli\u003eLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-\u0026Delta;\u0026Delta;CT method. Methods. 2001;25(4):402-8.\u003c/li\u003e\n \u003cli\u003eAl-dabbagh, Y. A., Rasheed, B. Y. and AL-Jumaa, Z. M. (2021). Molecular diagnosis of Mycoplasma gallisepticum in turkey in Mosul city. Veterinary Practitioner, 22(1), 1-4.\u003c/li\u003e\n \u003cli\u003eAl-Jumaa, Z. M., Jaber, M. T., \u0026amp; Al-Doori, A. A. (2024). Molecular detection of Chlamydophila felis from conjunctiva of cats infected with conjunctivitis and upper respiratory disease. Open Veterinary Journal, 14(12), 3289. https://doi.org/10.5455/OVJ.2024.v14.i12.13\u003c/li\u003e\n \u003cli\u003ePapa, G., Borodavka, A., \u0026amp; Desselberger, U. (2021). Viroplasms: assembly and functions of rotavirus replication factories. Viruses, 13(7), 1349.\u003c/li\u003e\n \u003cli\u003eZyara, A. M., Aldoori, A. A., Samawi, F. T., Kadhim, S. I., \u0026amp; Ali, Z. A. (2023). A relationship study of coronavirus (COVID-19) infection, blood groups, and some related factors in Iraqi patients. Baghdad Science Journal, 20(4), 32. https://doi.org/10.21123/bsj.2023.8871\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"miR-7, Rotavirus, Stool samples, Biomarker, Gene expression, NSP5, Diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-8058136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8058136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eVaccination is still the only effective way to prevent acute diarrhea in children under five, which is primarily caused by rotavirus. It is essential to look into virus-host interactions and find new biomarkers for early diagnosis in the absence of targeted treatments. The relationship between rotavirus infection and microRNA-7 (miR-7) expression is investigated in this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e 130 stool samples from children with acute gastroenteritis under the age of five were gathered. A commercial rapid antigen test was first used to screen for rotavirus infection, yielding 27 positive and 103 negative samples. All samples had their total RNA extracted, and RT-qPCR was used to measure the expression levels of miR-7, using miR-16 as an endogenous control. Receiver Operating Characteristic (ROC) curve analysis was used to evaluate miR-7's diagnostic performance.\u003c/p\u003e\n\u003cp\u003eAdditionally, the rotavirus NSP5 and NSP3 genes were found in a subset of samples using traditional RT-PCR. Findings: In stools from the rapid-test-positive group (n = 27) compared to the negative disease-control group (n = 103), MiR-7 expression was significantly upregulated (p \u0026lt; 0.0001). MiR-7 has a high diagnostic accuracy for differentiating these groups, according to ROC curve analysis, with an area under the curve (AUC) of 0.965, 85% sensitivity, and 99% specificity at an ideal cut-off of 5.83-fold change. Our particular RT-PCR assays detected the NSP5 gene in only 4 samples (3.1%) and the NSP3 gene in 1 sample (0.76%), despite the rapid test showing a positivity rate of 20.8%. This suggests a significant discrepancy that is probably caused by high primer specificity for specific rotavirus strains that are not prevalent in our cohort.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Children with gastroenteritis who test positive for rotavirus using a rapid antigen test have significantly higher levels of fecal miR-7. In this situation, it exhibits high diagnostic accuracy. It is a promising non-invasive biomarker due to its stability in stool. Future research employing more comprehensive molecular confirmation techniques (such as RT-qPCR targeting conserved genes) is necessary to conclusively establish miR-7's specificity for rotavirus infection, as the low confirmation rate by our particular RT-PCR assays indicates the association is with the rapid test status.\u003c/p\u003e","manuscriptTitle":"Molecular detection of NSP-5 and its relation with mi-RNA from patient infected with Rotavirus in Baghdad City","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-20 16:01:50","doi":"10.21203/rs.3.rs-8058136/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1461c5ca-151b-42ce-be0d-45a983cc1440","owner":[],"postedDate":"November 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-04T09:54:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-20 16:01:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8058136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8058136","identity":"rs-8058136","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}