Analysis Of Salivary Herpesviruses Reveals Associations Between HHV-6 And Long COVID Severity

Background Reactivation of human herpesviruses (HHVs), particularly EBV, is associated with more severe acute SARS-CoV-2 infections and the development of Long COVID (LC). Observations of higher anti-EBV antibody levels in individuals with LC support the idea that chronic reactivation of HHVs could contribute to LC pathology. HHV shedding in saliva has also been previously associated with saliva hormone levels. This study aims to examine the relationship between salivary shedding of HHV DNA and LC symptoms, as well as cortisol, testosterone, and estradiol levels. Methods We 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 minutes, and 8 and 16 hours after waking, across two consecutive days. Salivary cortisol, testosterone and estradiol were measured, and extracted nucleic acid was tested for EBV, HSV 1/2, HCMV and HHV-6 A/B using multiplex qPCR, plus SARS-CoV-2 and RNaseP using RT-qPCR. Findings Detection of salivary EBV and HHV-6 DNA was highest early in the morning. There were no significant differences in salivary cortisol, testosterone, or estradiol, or in EBV or HHV-6 shedding between the LC and control groups. However, salivary HHV-6 DNA levels were positively associated with a greater aggregated LC propensity score, as well as anxiety and depression scores. Interpretation The observed correlation between salivary HHV-6 shedding and symptom severity suggests HHV-6 may contribute to post-acute disease, though mechanisms remain unclear. While our study did not identify a relationship between salivary EBV shedding and LC, EBV may still play a role at earlier time points in the disease course, or in compartments not sampled here. 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. ### Competing Interest Statement A. I. co-founded RIGImmune, Xanadu Bio, Rho Bio, and PanV, and is a member of the Board of Directors of Roche Holding Ltd and Genentech. All other authors have no conflicts of interest. ### Funding Statement This work was supported in part by the Else Kroner Fresenius Prize for Medical Research 2023 (to A.I.), the Howard Hughes Medical Institute Collaborative COVID-19 Initiative (to A.I.), and the Howard Hughes Medical Institute (to A.I.), the Polybio Research Foundation (to D.P.) and the Steven and Alexandra Cohen Foundation (to D.P.). National Institutes for Health (NIH) funding sources supported AT (F31AI181508), SB (NHLBI T32HL007974-24) and CM (NCATS TL1 TR001864). Contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Mount Sinai Program for the Protection of Human Subjects (IRB 20-01758) and the Yale Institutional Review Board (IRB 2000029451) gave ethical approval for this work. Written informed consent was obtained from all enrolled participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The pre-specified rubric used for symptom survey encoding, the qPCR analysis pipeline, statistical model results, and relevant de-identified data included in our analyses will be included as supplementary files and/or deposited on GitHub upon publication of the peer-reviewed manuscript.