Associations between salivary HHV-6 and long COVID severity in a cross-sectional, case–control analysis

BACKGROUND: Reactivation of human herpesviruses (HHVs), particularly EBV, is associated with more severe acute SARS-CoV-2 infection and the development of Long COVID (LC). Reactivation of HHVs has been previously implicated in hormonal dysregulation, which has also been observed in LC, suggesting a potential link. This study aims to examine the relationship between LC symptoms and salivary HHV DNA, cortisol, testosterone, and oestradiol levels. METHODS: Our cross-sectional, case-control study enrolled 45 participants with LC, and 45 age-sex-matched controls. Surveys and validated health questionnaires were used to collect demographics, medical history, and symptom profiles. Saliva was self-collected at waking, 15, 30, and 45 min, and 8 and 16 h after waking, across two consecutive days. Salivary cortisol, testosterone and oestradiol were measured, and extracted nucleic acid was tested for EBV, HSV 1/2, HCMV and HHV-6 A/B using multiplex qPCR, and for SARS-CoV-2 and RNaseP using RT-qPCR. FINDINGS: Detection of salivary EBV and HHV-6 DNA copies was highest early in the morning. There were no significant differences in salivary cortisol, testosterone, or oestradiol, or in EBV or HHV-6 DNA between the LC and control groups. However, salivary HHV-6 DNA levels were positively associated with a greater aggregated LC propensity score (r = 0.24, 95% CI = 0.02-0.44, p = 0.015, adj. p = 0.030), as well as anxiety (β = 0.69, 95% CI = 0.02-1.36, p = 0.045) and depression (β = 0.24, 95% CI = 0.05-0.42, p = 0.013) scores. INTERPRETATION: The observed correlation between salivary HHV-6 DNA and symptom severity suggests HHV-6 may contribute to post-acute disease, though mechanisms remain unclear. While we did not identify a relationship between salivary EBV DNA and LC, EBV may still play a role at earlier time points in the disease course, or in compartments not sampled in this study. These findings highlight the potential importance of HHV-6 in LC pathophysiology and underscore the need for longitudinal, multi-compartment studies of herpesvirus reactivation in LC. FUNDING: This work was supported in part by the Else Kröner Fresenius Prize for Medical Research 2023 (to AI), the Howard Hughes Medical Institute Collaborative COVID-19 Initiative (to AI), and the Howard Hughes Medical Institute (to AI), the Polybio Research Foundation (to AI and DP) and the Steven and Alexandra Cohen Foundation (to DP). National Institutes for Health (NIH) funding sources supported AT, SB and CM.