Abstract

Background: The human virome encompasses both pathogenic and commensal viruses, with small circular DNA viruses such as Anelloviruses and Redondoviruses (ReDoVs) increasingly recognized as stable constituents of the respiratory tract. The objective of this study was to determine the prevalence of Anelloviruses (TTV, TTMV, TTMDV) and ReDoVs in nasopharyngeal samples of individuals screened for SARS-CoV-2, and to evaluate their potential clinical and oral health associations.

Methods: A total of 412 samples were collected from patients referred to health centers affiliated with Iran University of Medical Sciences (IUMS) between March and August 2023. Demographic, clinical, and oral health data were obtained through structured interviews and institutional records. Viral DNA was extracted using the High Pure Viral Nucleic Acid Kit and quantified by NanoDrop spectrophotometry. ReDoVs were amplified by PCR targeting a conserved capsid/Rep intergenic region with specific primers, while Anelloviruses were detected through a two-stage PCR assay with universal and species-specific primers (TTV, TTMV, TTMDV). Amplicons were confirmed by agarose gel electrophoresis, and PCR efficiency was validated by serial dilutions and quantitative assays. Statistical analyses included chi-square, Fisher’s exact test, logistic regression, and ROC curve modeling to assess associations between viral positivity, clinical symptoms, and oral health indicators.

Results: Anelloviruses were prevalent. TTV was detected in 81.6%, TTMV in 47.8%, and TTMDV in 43.9% of samples. ReDoVs were present in 22.1% of individuals, whereas SARS-CoV-2 was detected in 26.5%. No significant associations were found between SARS-CoV-2 positivity and Anellovirus or ReDoV infections. Instead, ReDoV positivity showed a strong correlation with gum problems (p < 0.0001, Cramér’s V = 0.467), and TTMV was similarly associated with gum problems (p = 0.015). Intra-family correlations were notable, with co-detection patterns observed among TTV, TTMV, and TTMDV, as well as between ReDoVs and TTMV/TTMDV. Logistic regression identified fever as a strong predictor of SARS-CoV-2 infection and gum problems as a predictor of ReDoV detection. Models for Anelloviruses lacked predictive power.

Conclusion: These findings highlight the ubiquity of Anelloviruses and the distinct clinical association of ReDoVs with oral health indicators. The results underscore the complexity of the respiratory virome and its potential as a biomarker of host–microbe interactions.