Associations Between Salivary HHV-6 And Long COVID Severity in a Cross-Sectional, Case-Control Analysis
- 1Yale School of Medicine
Abstract
BackgroundReactivation 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.
MethodsOur 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 minutes, and 8 and 16 hours 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.
FindingsDetection 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 to 0.44, p= 0.015, adj. p= 0.030), as well as anxiety ({beta}= 0.69, 95% CI= 0.02 to 1.36, p= 0.045) and depression ({beta}= 0.24, 95% CI= 0.05 to 0.42, p= 0.013) scores.
InterpretationThe 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.
FundingThis 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 A.I. and D.P.) and the Steven and Alexandra Cohen Foundation (to D.P.). National Institutes for Health (NIH) funding sources supported AT (NIAID F31AI181508), SB (NHLBI T32HL007974-24) and CM (NCATS TL1 TR001864).
Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSAn estimated 400 million people have developed Long COVID, a debilitating disease in which symptoms persist after SARS-CoV-2 infection, potentially for years, affecting multiple organ systems. We searched PubMed for articles published from Jan 1, 2020, to Dec 31, 2025, using terms for long COVID (e.g., "long COVID", "PASC") combined with herpesviruses (e.g., "Epstein-Barr virus", "HHV-6") or hormones (e.g., "cortisol", "testosterone", "estradiol"), excluding reviews, editorials and comments, with additional relevant studies identified separately. From this, we identified multiple studies which found that reactivation of human herpesviruses (HHVs) during or shortly after acute SARS-CoV-2 infection is associated with an increased risk of developing Long COVID, although there were also studies showing no evidence of elevated HHV reactivation in Long COVID. Some studies found cortisol was lower in people with Long COVID, though others showed no differences. Separately, HHVs, particularly HHV-6 and EBV, have been implicated in hormonal dysregulation, suggesting there may be link between the two findings. However, most data were generated from single timepoint serum measurements of hormones or antibodies.
Added value of this studyOur study focused on individuals living with Long COVID for, on average, 2 years. We used saliva, which is easy to self-collect at multiple timepoints, to examine potential connections between HHV reactivation, hormonal disturbances, and Long COVID symptom severity. We collected and tested samples over two days for both HHVs and hormones and administered detailed health and symptom surveys.
We did not observe differences in hormone levels between people with Long COVID and controls, although there were some trends between salivary HHV levels and hormones. We also did not observe clear differences in salivary EBV, CMV, HHV-6 or HSV DNA levels between controls and people living with Long COVID. However, we observed that elevated salivary HHV-6 DNA was associated with worse Long COVID symptom severity and potentially higher anxiety and depression scores, though the latter findings require further confirmation.
Implications of all the available evidenceTaken together with previous studies, our findings suggest that while EBV may play a role in the pathogenesis of Long COVID, before, during or shortly after acute infection, HHV-6 may have an ongoing pathological role. However, it remains unclear whether HHV-6 causes or exacerbates Long COVID symptoms or is merely an indirect indicator. Further studies are needed to paint a conclusive picture regarding the relationships between hormone disruption, HHV reactivation, and Long COVID.
Related
- Associations of rumination and worry with pain and functional limitation in knee osteoarthritis : A cross-sectional study accounting for radiographic severity
- Association between ABO Blood Group and COVID-19 Pneumonia Severity: A Cross-Sectional Study from Ethiopia's First Designated COVID-19 Treatment Center
- Associations of vitamin status and T-lymphocyte subsets with tic severity in children: a cross-sectional study using the Yale Global Tic Severity Scale
- Breakfast Dietary Patterns and Their Associations with Metabolic Indicators and NAFLD Severity: A Cross-Sectional Study Among Iranian Adults.
- Nasal methicillin-resistant in adult atopic dermatitis: Severity and virulence associations—A cross-sectional study
BibTeX
@misc{laxton2026associations,
title = {{Associations Between Salivary HHV-6 And Long COVID Severity in a Cross-Sectional, Case-Control Analysis}},
author = {C. S. Laxton and A. Tabachnikova and L. Cooke and K. Wang and S. Blaser and J. Silva and J. Wood and H. Nam and Z. Lu and C. Miller and G. Rodrigues and V. Fisher and C. Guirgis and W. B. Hooper and A. Lee and M. Doerstling and B. Bhattacharjee and L. Guan and D. Putrino and A. Iwasaki},
howpublished = {medRxiv},
year = {2026},
month = may,
doi = {10.64898/2026.05.19.26353495},
url = {https://www.medrxiv.org/content/10.64898/2026.05.19.26353495v2},
note = {CC BY},
}
RIS
TY - UNPB
TI - Associations Between Salivary HHV-6 And Long COVID Severity in a Cross-Sectional, Case-Control Analysis
AU - C. S. Laxton
AU - A. Tabachnikova
AU - L. Cooke
AU - K. Wang
AU - S. Blaser
AU - J. Silva
AU - J. Wood
AU - H. Nam
AU - Z. Lu
AU - C. Miller
AU - G. Rodrigues
AU - V. Fisher
AU - C. Guirgis
AU - W. B. Hooper
AU - A. Lee
AU - M. Doerstling
AU - B. Bhattacharjee
AU - L. Guan
AU - D. Putrino
AU - A. Iwasaki
PY - 2026
DA - 2026/05/19
PB - medRxiv
DO - 10.64898/2026.05.19.26353495
UR - https://www.medrxiv.org/content/10.64898/2026.05.19.26353495v2
AB - BackgroundReactivation 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. MethodsOur 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 minutes, and 8 and 16 hours 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. FindingsDetection 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 to 0.44, p= 0.015, adj. p= 0.030), as well as anxiety ({beta}= 0.69, 95% CI= 0.02 to 1.36, p= 0.045) and depression ({beta}= 0.24, 95% CI= 0.05 to 0.42, p= 0.013) scores. InterpretationThe 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. FundingThis 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 A.I. and D.P.) and the Steven and Alexandra Cohen Foundation (to D.P.). National Institutes for Health (NIH) funding sources supported AT (NIAID F31AI181508), SB (NHLBI T32HL007974-24) and CM (NCATS TL1 TR001864). Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSAn estimated 400 million people have developed Long COVID, a debilitating disease in which symptoms persist after SARS-CoV-2 infection, potentially for years, affecting multiple organ systems. We searched PubMed for articles published from Jan 1, 2020, to Dec 31, 2025, using terms for long COVID (e.g., "long COVID", "PASC") combined with herpesviruses (e.g., "Epstein-Barr virus", "HHV-6") or hormones (e.g., "cortisol", "testosterone", "estradiol"), excluding reviews, editorials and comments, with additional relevant studies identified separately. From this, we identified multiple studies which found that reactivation of human herpesviruses (HHVs) during or shortly after acute SARS-CoV-2 infection is associated with an increased risk of developing Long COVID, although there were also studies showing no evidence of elevated HHV reactivation in Long COVID. Some studies found cortisol was lower in people with Long COVID, though others showed no differences. Separately, HHVs, particularly HHV-6 and EBV, have been implicated in hormonal dysregulation, suggesting there may be link between the two findings. However, most data were generated from single timepoint serum measurements of hormones or antibodies. Added value of this studyOur study focused on individuals living with Long COVID for, on average, 2 years. We used saliva, which is easy to self-collect at multiple timepoints, to examine potential connections between HHV reactivation, hormonal disturbances, and Long COVID symptom severity. We collected and tested samples over two days for both HHVs and hormones and administered detailed health and symptom surveys. We did not observe differences in hormone levels between people with Long COVID and controls, although there were some trends between salivary HHV levels and hormones. We also did not observe clear differences in salivary EBV, CMV, HHV-6 or HSV DNA levels between controls and people living with Long COVID. However, we observed that elevated salivary HHV-6 DNA was associated with worse Long COVID symptom severity and potentially higher anxiety and depression scores, though the latter findings require further confirmation. Implications of all the available evidenceTaken together with previous studies, our findings suggest that while EBV may play a role in the pathogenesis of Long COVID, before, during or shortly after acute infection, HHV-6 may have an ongoing pathological role. However, it remains unclear whether HHV-6 causes or exacerbates Long COVID symptoms or is merely an indirect indicator. Further studies are needed to paint a conclusive picture regarding the relationships between hormone disruption, HHV reactivation, and Long COVID.
ER -