Abstract
Coronavirus disease 2019 (COVID-19) remains a major global health concern, with emerging evidence highlighting the role of the human microbiome in influencing disease severity. While extensive research has been conducted on COVID-19, studies examining host-pathogen interactions at the transcriptomic level remain limited. In this study, we investigated the metatranscriptomic profiles of forty nasopharyngeal samples collected from COVID-19 patients across different Bangladeshi cohorts. Sequencing data were processed to analyze taxonomic composition, microbial diversity, and antimicrobial resistance gene (ARG) patterns using multiple bioinformatic pipelines. COVID-19 positive and asymptomatic patients exhibited a higher abundance of pathogenic and multidrug-resistant bacteria, whereas COVID-19 negative individuals showed increased fungal diversity. Differential gene expression analysis revealed significant upregulation of immune response related genes, including pro-inflammatory cytokines, in COVID-19 positive cases. Notably, asymptomatic patients demonstrated reduced TLR4 expression, suggesting a potential reducing of innate immune activation, which may contribute to asymptomatic clinical outcomes. Additionally, functional enrichment highlighted active ARG expression in positive cases, indicating potential links between the respiratory microbiome and host immune modulation. These findings provide insights into the host-microbiome interplay underlying COVID-19 severity and highlight the need for further validation in larger, ethnically diverse cohorts with comprehensive clinical metadata.
Data availability
All the data related to this manuscript is available in the supplement files and all the raw data is uploaded to NCBI under the accession number of PRJNA1298058. Formal permission was taken from Kanehisa laboratories to publish the result of using KEGG software, both in print and digital, under the CC BY 4.0 open access license.
References
Zhu, N. et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl. J. Med. 382, 727–733. https://doi.org/10.1056/nejmoa2001017 (2020).
Salata, C., Calistri, A., Parolin, C. & Palù, G. Coronaviruses: A paradigm of new emerging zoonotic diseases. Pathog. Dis. https://doi.org/10.1093/femspd/ftaa006 (2019).
Tan, W. et al. A novel coronavirus genome identified in a cluster of pneumonia cases - Wuhan, China 2019–2020. China CDC Wkly. 2, 61–62 (2020).
Du Toit, A. Outbreak of a novel coronavirus. Nat. Rev. Microbiol. 18, 123. https://doi.org/10.1038/s41579-020-0332-0 (2020).
Zhou, F. et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: A retrospective cohort study. Lancet 395, 1054–1062. https://doi.org/10.1016/S0140-6736(20)30566-3 (2020).
Kumar, A. et al. COVID-19 mechanisms in the human body—What we know so far. Front. Immunol. https://doi.org/10.3389/fimmu.2021.693938 (2021).
Daamen, A. R. et al. Comprehensive transcriptomic analysis of COVID-19 blood, lung, and airway. Sci. Rep. https://doi.org/10.1038/s41598-021-86002-x (2021).
Xiong, Y. et al. Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients. Emerg. Microbes Infect. 9, 761–770. https://doi.org/10.1080/22221751.2020.1747363 (2020).
Hoque, M. N. et al. Diversity and genomic determinants of the microbiomes associated with COVID-19 and non-COVID respiratory diseases. Gene Rep. 23, 101200. https://doi.org/10.1016/j.genrep.2021.101200 (2021).
Chen, N. et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: A descriptive study. Lancet 395, 507–513. https://doi.org/10.1016/S0140-6736(20)30211-7 (2020).
Hoque, M. N. et al. SARS-CoV-2 infection reduces human nasopharyngeal commensal microbiome with inclusion of pathobionts. Sci. Rep. 11, 24042. https://doi.org/10.1038/s41598-021-03245-4 (2021).
Avraham, R. et al. A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes. Nat. Protoc. 11, 1477–1491. https://doi.org/10.1038/nprot.2016.090 (2016).
Zhang, H. et al. Metatranscriptomic characterization of Coronavirus Disease 2019 identified a host transcriptional classifier associated with immune signaling. Clin. Infect. Dis. 73, 376–385. https://doi.org/10.1093/cid/ciaa663 (2021).
Haiminen, N., Utro, F., Seabolt, E. & Parida, L. Functional profiling of COVID-19 respiratory tract microbiomes. Sci. Rep. https://doi.org/10.1038/s41598-021-85750-0 (2021).
Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat. Methods 6, 377–382. https://doi.org/10.1038/nmeth.1315 (2009).
Li, Y., Samuvel, D. J., Sundararaj, K. P., Lopes-Virella, M. F. & Huang, Y. IL‐6 and high glucose synergistically upregulate MMP‐1 expression by U937 mononuclear phagocytes via ERK1/2 and JNK pathways and c‐Jun. J. Cell. Biochem. 110, 248–259. https://doi.org/10.1002/jcb.22532 (2010).
Kanehisa, M., Furumichi, M., Sato, Y., Matsuura, Y. & Ishiguro-Watanabe, M. KEGG: Biological systems database as a model of the real world. Nucleic Acids Res. 53, D672–D677. https://doi.org/10.1093/nar/gkae909 (2025).
Kanehisa, M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28, 1947–1951. https://doi.org/10.1002/pro.3715 (2019).
Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30. https://doi.org/10.1093/nar/28.1.27 (2000).
Waldman, A. J. & Balskus, E. P. The Human Microbiota, Infectious Disease, and Global Health: Challenges and opportunities. ACS Infect. Dis. 4, 14–26. https://doi.org/10.1021/acsinfecdis.7b00232 (2018).
Wang, B., Yao, M., Lv, L., Ling, Z. & Li, L. The human microbiota in health and disease. Engineering 3, 71–82. https://doi.org/10.1016/j.eng.2017.01.008 (2017).
Karkman, A., Lehtimäki, J. & Ruokolainen, L. The ecology of human microbiota: Dynamics and diversity in health and disease. Ann. N. Y. Acad. Sci. 1399, 78–92. https://doi.org/10.1111/nyas.13326 (2017).
Cho, S. I., Yoon, S. & Lee, H.-J. Impact of comorbidity burden on mortality in patients with COVID-19 using the Korean health insurance database. Sci. Rep. https://doi.org/10.1038/s41598-021-85813-2 (2021).
Osibogun, A. et al. Outcomes of COVID-19 patients with comorbidities in southwest Nigeria. PLOS ONE 16, e0248281. https://doi.org/10.1371/journal.pone.0248281 (2021).
Yin, T., Li, Y., Ying, Y. & Luo, Z. Prevalence of comorbidity in Chinese patients with COVID-19: Systematic review and meta-analysis of risk factors. BMC Infect. Dis. https://doi.org/10.1186/s12879-021-05915-0 (2021).
Lopez, S. M. C. et al. A method of processing nasopharyngeal swabs to enable multiple testing. Pediatr. Res. 86, 651–654. https://doi.org/10.1038/s41390-019-0498-1 (2019).
Peng, J. et al. Fungal co-infection in COVID-19 patients: Evidence from a systematic review and meta-analysis. Aging 13, 7745–7757. https://doi.org/10.18632/aging.202742 (2021).
Verweij, P. E. et al. Diagnosing COVID-19-associated pulmonary aspergillosis. Lancet Microbe 1, e53–e55. https://doi.org/10.1016/S2666-5247(20)30027-6 (2020).
Canning, B., Senanayake, V., Burns, D., Moran, E. & Dedicoat, M. Post-influenza aspergillus ventriculitis. Clin. Infect. Pract. 7–8, 100026. https://doi.org/10.1016/j.clinpr.2020.100026 (2020).
González, R. & Elena, S. F. The interplay between the host microbiome and pathogenic viral infections. mBio https://doi.org/10.1128/mbio.02496-21 (2021).
Palleja, A. et al. Recovery of gut microbiota of healthy adults following antibiotic exposure. Nat. Microbiol. 3, 1255–1265. https://doi.org/10.1038/s41564-018-0257-9 (2018).
Zuo, T. et al. Alterations in gut microbiota of patients with COVID-19 during time of hospitalization. Gastroenterology 159, 944-955.e8. https://doi.org/10.1053/j.gastro.2020.05.048 (2020).
Umeda, L. et al. Immuno-microbial signature of vaccine-induced immunity against SARS-CoV-2. Vaccines 12, 637. https://doi.org/10.3390/vaccines12060637 (2024).
Ng, D. L. et al. A diagnostic host response biosignature for COVID-19 from RNA profiling of nasal swabs and blood. Sci. Adv. 7, eabe5984. https://doi.org/10.1126/sciadv.abe5984 (2021).
Hyblova, M. et al. Metatranscriptome analysis of nasopharyngeal swabs across the varying severity of COVID-19 disease demonstrated unprecedented species diversity. Microorganisms 11, 1804. https://doi.org/10.3390/microorganisms11071804 (2023).
Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506. https://doi.org/10.1016/s0140-6736(20)30183-5 (2020).
Liu, Y. et al. Elevated plasma levels of selective cytokines in COVID-19 patients reflect viral load and lung injury. Natl. Sci. Rev. 7, 1003–1011. https://doi.org/10.1093/nsr/nwaa037 (2020).
Nuñez, G. & Clarke, M. F. The Bcl-2 family of proteins: Regulators of cell death and survival. Trends Cell. Biol. 4, 399–403. https://doi.org/10.1016/0962-8924(94)90053-1 (1994).
Lin, Z. et al. Airway microbiota and immunity associated with chronic obstructive pulmonary disease severity. J. Transl. Med. 23, 962. https://doi.org/10.1186/s12967-025-06986-2 (2025).
Zhang, E. et al. Immune-related gene-based novel subtypes to establish a model predicting the risk of prostate cancer. Front. Genet. 11, 595657. https://doi.org/10.3389/fgene.2020.595657 (2020).
Daniel, N., Lécuyer, E. & Chassaing, B. Host/microbiota interactions in health and diseases—Time for mucosal microbiology! Mucosal Immunol. 14, 1006–1016. https://doi.org/10.1038/s41385-021-00383-w (2021).
Tian, W. et al. Immune suppression in the early stage of COVID-19 disease. Nat. Commun. https://doi.org/10.1038/s41467-020-19706-9 (2020).
Fulzele, S. et al. COVID-19 virulence in aged patients might be impacted by the host cellular microRNAs abundance/profile. Aging Dis. 11, 509–522. https://doi.org/10.14336/AD.2020.0428 (2020).
Tang, Y. et al. Cytokine storm in COVID-19: The current evidence and treatment strategies. Front. Immunol. 11, 1708. https://doi.org/10.3389/fimmu.2020.01708 (2020).
Tjan, L. H. et al. Early differences in cytokine production by severity of Coronavirus Disease 2019. J. Infect. Dis. 223, 1145–1149. https://doi.org/10.1093/infdis/jiab005 (2021).
Coperchini, F. et al. The cytokine storm in COVID-19: Further advances in our understanding the role of specific chemokines involved. Cytokine Growth Factor Rev. 58, 82–91. https://doi.org/10.1016/j.cytogfr.2020.12.005 (2021).
Cui, J., Chen, Y., Wang, H. Y. & Wang, R.-F. Mechanisms and pathways of innate immune activation and regulation in health and cancer. Hum. Vaccines Immunother. 10, 3270–3285. https://doi.org/10.4161/21645515.2014.979640 (2014).
Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. 21, 335–376. https://doi.org/10.1146/annurev.immunol.21.120601.141126 (2003).
Sahanic, S. et al. SARS-CoV-2 activates the TLR4/MyD88 pathway in human macrophages: A possible correlation with strong pro-inflammatory responses in severe COVID-19. Heliyon 9, e21893. https://doi.org/10.1016/j.heliyon.2023.e21893 (2023).
Ziegler, C. G. K. et al. SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues. Cell 181, 1016-1035e.e19. https://doi.org/10.1016/j.cell.2020.04.035 (2020).
Wang, L., Wang, S. & Li, W. RSeQC: Quality control of RNA-seq experiments. Bioinforma. Oxf. Engl. 28, 2184–2185. https://doi.org/10.1093/bioinformatics/bts356 (2012).
Dobin, A. et al. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. https://doi.org/10.1093/bioinformatics/bts635 (2013).
McMurdie, P. J., Holmes, S. & Watson, M. Phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8, e61217. https://doi.org/10.1371/journal.pone.0061217 (2013).
Oksane, J. et al. The vegan package. Community Ecol. package. 10, 631–637 (2007).
Kassambara, A. & ggpubr ggplot2 Based Publication Ready Plots. (2025). Available: https://rpkgs.datanovia.com/ggpubr/
Kalantar, K. L. et al. IDseq—An open source cloud-based pipeline and analysis service for metagenomic pathogen detection and monitoring. Gigascience https://doi.org/10.1093/gigascience/giaa111 (2020).
Gu, Z., Gu, L., Eils, R., Schlesner, M. & Brors, B. Circlize implements and enhances circular visualization in R. Bioinforma. Oxf. Engl. 30, 2811–2812. https://doi.org/10.1093/bioinformatics/btu393 (2014).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550. https://doi.org/10.1186/s13059-014-0550-8 (2014).
Luo, W. & Brouwer, C. Pathview: An R/Bioconductor package for pathway-based data integration and visualization. Bioinforma. Oxf. Engl. 29, 1830–1831. https://doi.org/10.1093/bioinformatics/btt285 (2013).
Acknowledgements
The authors would like to acknowledge National Institute of Laboratory Medicine & Referral Center (NILMRC), Dhaka, Bangladesh for the support during sample collection. The authors also acknowledge Bangladesh Council of Scientific and Industrial Research (BCSIR), Bangladesh and Ministry of Science and Technology, Bangladesh for the funding and infrastructure support.
Funding
This work has been funded by the Ministry of Science and Technology, Bangladesh.
Author information
Authors and Affiliations
Contributions
Experimental Design: Murshed Hasan Sarkar, Sanjana Fatema Chowdhury, Salim Khan Sample Collection: Md. Maruf Ahmed Molla, Tasnim Nafisa, Mahmuda Yeasmin, Asish Kumar Ghosh Library Preparation and Sequencing: Murshed Hasan Sarkar, Md. Saddam Hossain, Iffat Jahan Data Analysis and Primary Analysis: Sanjana Fatema Chowdhury, Murshed Hasan Sarkar, Syed Muktadir Al Sium, Tanay Chakrovarty Manuscript Preparation: Sanjana Fatema Chowdhury, Murshed Hasan Sarkar, Syed Muktadir Al Sium, Showti Raheel Naser Logistic Support: Md. Ahashan Habib, Shahina Akter, Tanjina Akhtar Banu, Barna Goswami.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethical approval and consent to participate
The ethical permission of the protocol for sample collection from patients, sample processing, and other consecutive laboratory work was taken from the National Institute of Laboratory Medicine and Referral Center (NILMRC) of Bangladesh.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Chowdhury, S.F., Sarkar, M.H., Al Sium, S.M. et al. Metatranscriptomic insights into host-microbiome interactions underlying asymptomatic COVID-19 cases. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40563-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-40563-x