Chronic fatigue syndrome, depression, and anxiety symptoms due to relapsing-remitting multiple sclerosis are associated with reactivation of Epstein-Barr virus and Human Herpesvirus 6

Demographic and clinical features of RRMS patients and controls. Table 1 shows the demographic and clinical data of the RRMS patients and normal controls in this study. We found no differences observed in sex, age, BMI, education, and marital status between both study groups. There were somewhat more smokers among normal volunteers. All neuropsychiatric rating scale scores were significantly higher in RRMS patients than in controls. Construction of principal components Table 2 shows the outcome of different PCAs. First, we checked whether one PC could be extracted from the total FF, HAMD, and HAMA scores (see PCA #1). The first PC explained 90.01% of the variance and all loadings were higher than 0.934 (labeled PC FFAD from fibro- fatigue, anxiety, and depression). In PCA #2, we added the MSSS and EDSS scores. We were again able to extract one PC from these 5 scores which explained 84.88% of the variance, while all 5 variables showed high loadings (all > 0.870). This PC was labeled PC DISFFAD from disabilities, fibro-fatigue, anxiety, and depression. We were able to extract PCs, which complied with the a priori quality criteria, from the three IgG/IgA/IgM-EBNA-1 values (KMO=0.672, explained variance is 72.83%, labeled PC EBNA), three HHV-6 dUTPses (KMO=0.725, explained variance is 77.64%, labeled PC HHV-6-dUTPase), and three EBV dUTPases (KMO=0.675, explained variance is 73.34%, PC EBV-6-dUTPase). Consequently, we . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 13 investigated whether one PC could be extracted from the three neuropsychiatric scores, EDSS, MSSS and the three viral PCs. Table 2, PCA #3 shows that one adequate PC could be extracted from these 8 variables with a good KMO value and explaining 75.58% of the variance. All eight variables loaded highly on this first PC and all loadings were higher than 0.800. With the exception of IgG-dUTPase (r=-0.300, p=0.026, n=55, without false discovery rate p correction for multiple comparisons), no significant point-biserial correlations were observed between natalizumab administration and any viral data within the restricted cohort of RRMS patients. The administration of beta-interferon-1 β was significantly correlated (without p- correction) with decreased levels of IgM-EBNA (r=-0.283, p=0.036), IgA-EBNA (r=-0.336, p=0.012), IgM-EBV-dUTPase (r=-0.305, p=0.023), and IgA-HHV-6-dUTPase (r=-0.267, p=0.049). However, all significances were nullified through the application of FDR p correction. Correlations between clinical ratings and immune responses to viral antigens In all subjects combined, we found strong associations between PC DISFADD and the three PC scores (PC EBNA, PC EBV-dUTPases, and PC HHV-6-dUTPases), and the IgG/IgA/IgM responses to EBNA, EBV-dUTPases, and HHV-6-dUTPases (see Table 3). In the restricted RRMS patient sample, we found significant correlations between PC DISFFAD and the three PC scores, IgM/IgA-EBV-dUTPase, and IgA directed to HHV-6-dUTPase and EBNA. Consequently, we have also examined the associations between the three constructed “viral” PCs and the relevant rating scale scores ( Table 4). We found significant correlations between the three viral PCs and the total FF, total HAMD, total HAMA scores, pure FF and physiosomatic scores, and pure HAMD and HAMA scores. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 14 Immune responses to EBV and HHV-6 predict severity of neuropsychiatric symptoms. Table 5 shows the results of multivariable regression analyses with the neuropsychiatric rating scale scores as dependent variables and IgG/IgA/IgM reactivity to viral antigens as explanatory variables while allowing for the effects of age, sex, BMI, and smoking. Nevertheless, these possible confounding variables were always non-significant. We found that IgG/IgM-HHV- 6-dUTPAses explained together 63.7% of the variance in PC DISFADD in the total study group. IgG/IgM-HHV-6-dUTPAses significantly predicted the total scores of the three neuropsychiatric rating scales. IgG-EBV-dUTPase was additionally associated with the total HAMA score. A large part (38.7% - 51.0%) of the variance in the pure FF, physiosomatic, HAMD and HAMA scores was predicted by a combination of the viral reactivation biomarkers. The pure FF score was best predicted by IgG/IgM-HHV-6-dUTPases, whereas the pure HAMD score was predicted by IgG/IgM-EBV-dUTPase. Figure 1 shows the partial regression of the pure FF score on IgG directed against HHV /i2 6-dUTPase. Figure 2 shows the partial regression of the pure HAMD score on IgG-EBV-dUTPase. In the restricted study group of RRMS patients, we found that 16.1% of the variance in PC DISFFAD was explained by the regression on IgA-HHV-6-dUTPase (F=10.18, df=1/53, p=0.002, β =0.401). Figure 3 shows the partial regression of PC DISFADD on IgA-HHV-6-dUTPase (after adjusting for sex and age).

Increased chronic fatigue, depression, and anxiety symptoms in RRMS. The first finding of this study indicates that RRMS is marked by an exacerbation of chronic fatigue syndrome symptoms, alongside heightened symptoms of depression and anxiety. Nevertheless, the intensity of these three symptom domains is elevated only to a moderate extent. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 15 Consequently, the average HAMD score among patients diagnosed with RRMS was recorded at 11.7 (SD=3.5). In contrast, outpatients exhibiting very mild MDD presented HAMD scores ranging from 15.2 (SD=6.3) to 17.7 (SD=6.5) (Vasupanrajit et al., 2024). In inpatients diagnosed with MDD, the average score on the HAMD is significantly elevated, specifically at 21.3 (SD=3.7) (Maes et al., 1986). In the current investigation, the average HAMA score was recorded at 13.5 (SD=3.6). In contrast, individuals diagnosed with MDD exhibit scores ranging from 27.2 (SD=2.6) to 29.5 (SD=1.8) (Maes et al., 1994). Furthermore, subjects experiencing minor depression present with a mean score of 15.6 (SD=1.4) (Maes et al., 1994). A similar pattern can be discerned in relation to the FF scale score. In our cohort of patients diagnosed with RRMS, the mean score on the FF scale was recorded at 23.6, with a standard deviation of 4.7. In individuals presenting with CFS/ME, the FF score is significantly higher, averaging approximately 43.3 (SD=7.7) (Maes et al., 2012). Nonetheless, the HAMD, HAMA, and FF scores obtained in patients with RRMS are either higher or comparable to the mean scores recorded in individuals experiencing a stable phase of schizophrenia, specifically ranging from 4.3 to 9.9, 5.3 to 16.3, and 5.8 to 18.2, respectively (Kanchanatawan et al., 2017). The significance of these affective and FF scores cannot be overstated, as they serve as the primary predictors of diminished quality of life among individuals diagnosed with schizophrenia (Kanchanatawan et al., 2019). It is noteworthy that the intensity of affective and chronic fatigue symptoms may escalate during acute relapses (Hanken et al., 2019; Khatibi et al., 2020; McCabe, 2005). However, it is also important to consider that some authors have documented non- significant associations between affective symptoms and the various clinical phases (Jefferies and Lambon Ralph, 2006). . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 16 It is crucial to note that the current study computed sub scores that evaluated pure cognitive depressive and anxiety symptoms in isolation from chronic fatigue and physiosomatic symptoms. Furthermore, given that we evaluated the HAMD, HAMA, and FF scales, as well as their cognitive and physiosomatic subdomains, within the three months preceding the study, it is reasonable to infer that the remitted phase of RRMS is associated with chronically elevated ratings of chronic fatigue syndrome, physiosomatic and affective symptoms. Given that patients with lifetime diagnoses of MDD, as well as CFS/ME, were excluded, it is reasonable to infer that RRMS is defined by new onset chronic fatigue syndrome and affective symptoms. The current study reveals a significant finding: a singular latent construct can be derived from the affective and chronic fatigue symptoms, as well as the EDSS and MSSS scores. This suggests that the interconnected relationships between heightened motor and sensory impairments, along with increased chronic fatigue and affective symptoms, represent highly interrelated manifestations of RRMS. This further emphasizes that these symptoms and disabilities exhibit shared underlying pathways. Neuropsychiatric scores are predicted by viral replication. The second significant discovery of this study is the significant correlation between PC DISFFAD, the severity of depression, anxiety, and chronic fatigue symptoms, and the markers of HHV-6 and EBV reactivation, replication, or active "abortive" infection (Sommer et al., 1996; Tiwari et al., 2022) we measured in our study. In addition, we were able to extract a single latent construct from the viral reactivation and replication biomarkers, as well as the disability, affective, and chronic fatigue scores. This not only demonstrates a strong correlation between the . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 17 viral biomarkers, disabilities, and neuropsychiatric scores in RRMS, but also indicates that HHV-6 and EBV reactivation/replication are critical components of this disorder. Prior research indicates that FFAD symptoms during acute relapses may be ascribed to activated immune-inflammatory and oxidative stress pathways (Moore et al., 2012; Morris and Maes, 2013a; Ormstad et al., 2020; Šabanagi ć -Hajrić et al., 2015). For example, elevated levels of IL-6 are positively correlated with the depressive symptoms associated with RRMS (Kallaur et al., 2016; Koutsouraki et al., 2011). Kallaur et al. (2016) demonstrated that depression is a manifestation of the neurological impairments associated with multiple sclerosis and that both symptom categories are predicted by indicators of peripheral immunological activation (Kallaur et al., 2016). In the relapsing phase of RRMS, FFAD symptoms are significantly correlated with several inflammatory and anti-inflammatory cytokines, particularly those of the T helper (Th)17 axis (Almulla et al., 2023a). Consequently, it was determined that the activation of immune- inflammatory, autoimmune, and oxidative pathways, along with the resultant damage to CNS structures in MS, may heighten susceptibility to affective symptoms and chronic fatigue syndrome (de Carvalho Jennings Pereira et al., 2020; Feinstein, 2004; Morris and Maes, 2013a). It is essential to emphasize that affective disorders, CFS/ME are marked by activated immune- inflammatory and oxidative stress pathways (Maes, 2023; Maes and Carvalho, 2018; Morris and Maes, 2013b). Consequently, the hypothesis posits that heightened neurotoxicity resulting from immune activation (e.g., elevated M1 macrophages, Th1 and Th17 cytokines) in RRMS may impair affective circuits and energy homeostasis in the CNS, consequently leading to affective symptoms, chronic fatigue, and psychosomatic manifestations (Maes and Carvalho, 2018; Morris and Maes, 2013a). It is crucial to note that dUTPases may function as PAMPs and activate Toll- . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 18 Like receptors, nuclear factor- κ B, and M1 and Th-1 cytokine production, thereby exacerbating the pathogenesis of MS (Ariza et al., 2022; Parroche, 2011; Waldman et al., 2008; Williams et al., 2016; Williams et al., 2023; Zhang et al., 2017). HHV-6 seropositivity and reactivation are linked to altered levels of pro-inflammatory cytokines, including Th1 cytokines (Chapenko et al., 2003). Additionally, autoimmune responses and the resulting tissue injury observed in RRMS may be exacerbated by the reactivation and replication of EBV and HHV-6, particularly through cross-reactivity or mimicry (Fotheringham and Jacobson, 2005; Lanz et al., 2022). Consequently, HHV-6 and EBV reactivation may not only sustain ongoing immune activation during the remission phase of RRMS (Almulla et al., 2024) but also appear to drive the affective symptoms and chronic fatigue syndrome associated with RRMS.

The findings of the present investigation warrant replication in other nations and cultures. The results could have been more intriguing if we had also evaluated the viral biomarkers and neuropsychiatric rating instruments during acute relapses. In order to evaluate biomarkers and rating scales during the acute relapse phase and remission, future research should employ a prospective design. Although some people may consider that the study was performed using a smaller study sample, the sample size was estimated a priori based on a power of 0.8. Moreover, the power of 1.0 was obtained through the post-hoc computation of the achieved power in the primary statistical analysis, which involved multiple regression with PC DISFADD as the dependent variable and the viral biomarkers as explanatory variables.

. CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 19 This study demonstrates a significant association between heightened IgG/IgA/IgM reactivity to EBNA, EBV-dUTPase, and HHV-6 dUTPase with PC DISFFAD, as well as the severity of depression, anxiety, and chronic fatigue syndrome in the context of RRMS. A significant portion of depression and anxiety symptoms, which are distinct from psychosomatic symptoms (38.3%-38.7%), as well as psychosomatic symptoms themselves (51 %), is anticipated to be influenced by immune reactivity to HHV-6-dUTPase and EBV-dUTPase. The findings suggest that the reactivation, replication, or active "abortive" infection of HHV-6 and EBV plays a significant role in the initiation or persistence of disabilities, affective symptoms, and chronic fatigue syndrome associated with RRMS.

The authors are deeply grateful to the Neuroscience Center of Alsader Medical City in Al-Najaf province, Iraq, for their invaluable support in the data collection process. Ethical approval and consent to participate. The Ethics Committee of the College of Medical Technology at the Islamic University of Najaf, Iraq, granted sanction for the investigation (Document No. 11/2021). Written informed consent was obtained from all patients and control participants, and all procedures were conducted in accordance with Iraqi and international ethical standards. Declaration of interest No conflicts of interest are disclosed by the authors. Funding . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 20 AFA received funding for the project from the C2F program at Chulalongkorn University in Thailand, with grant number 64.310/436/2565. The Thailand Science Research, and Innovation Fund at Chulalongkorn University (HEA663000016) and the Sompoch Endowment Fund (Faculty of Medicine) MDCU (RA66/016) provided funding to MM. Immunosciences Lab., Inc., Los Angeles, CA, USA, and Cyrex Labs, LLC, Phoenix, AZ, USA, provided funding for the execution of all antibody assays. Author’s contributions AFA oversaw the blood sample collection and patient-related procedures. AV and AFA conducted the serum biomarker quantification. The statistical analysis was conducted by MM. Visualization was executed by MM. MM authored the initial manuscript, which was subsequently revised by AFA, EV, ED, DS, YZ, and AV. All authors authorized the most recent version. Availability of data The corresponding author (MM) is prepared to grant access to the file associated with this study upon receipt of a valid request and subsequent to a comprehensive analysis of the data set.

. CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 21 Abbasi, M., Nabavi, S.M., Fereshtehnejad, S.M., Ansari, I., Zerafatjou, N., Shayegannejad, V ., Mohammadianinejad, S.E., Farhoudi, M., Noorian, A., Razazian, N., Abedini, M., Faraji, F., 2016. Risk factors of Multiple sclerosis and their Relation with Disease Severity: A Cross- sectional Study from Iran. Arch Iran Med 19 (12), 852-860. Al-Hadrawi, D.S., Al-Rubaye, H.T., Almulla, A.F., Al-Hakeim, H.K., Maes, M., 2023. Lowered oxygen saturation and increased body temperature in acute COVID-19 largely predict chronic fatigue syndrome and affective symptoms due to Long COVID: A precision nomothetic approach. Acta Neuropsychiatr 35 (2), 76-87. Al-Hakeim, H.K., Al-Musawi, A.F., Al-Mulla, A., Al-Dujaili, A.H., Debnath, M., Maes, M., 2022. The interleukin-6/interleukin-23/T helper 17-axis as a driver of neuro-immune toxicity in the major neurocognitive psychosis or deficit schizophrenia: A precision nomothetic psychiatry analysis. PLoS One 17 (10), e0275839. Almulla, A.F., Abdul Jaleel, A.K., Abo Algon, A.A., Tunvirachaisakul, C., Hassoun, H.K., Al- Hakeim, H.K., Maes, M., 2023a. Mood Symptoms and Chronic Fatigue Syndrome Due to Relapsing-Remitting Multiple Sclerosis Are Associated with Immune Activation and Aberrations in the Erythron. Brain Sci 13 (7). Almulla, A.F., Al-Rawi, K.F., Maes, M., Al-Hakeim, H.K., 2021. In schizophrenia, immune- inflammatory pathways are strongly associated with depressive and anxiety symptoms, which are part of a latent trait which comprises neurocognitive impairments and schizophrenia symptoms. J Affect Disord 287, 316-326. Almulla, A.F., Maes, M., Zhou, B., Al-Hakeim, H.K., V ojdani, A., 2023b. Brain-targeted autoimmunity is strongly associated with Long COVID and its chronic fatigue syndrome as well as its affective symptoms. medRxiv, 2023.2010.2004.23296554. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 22 Almulla, A.F., V ojdani, A., Zhang, Y ., V ojdani, E., Maes, M., 2024. Reactivation of Human Herpesvirus 6 and Epstein-Barr Virus in relapsing remitting multiple sclerosis: association with disabilities, disease progression, and inflammatory processes. medRxiv, 2024.2009.2010.24313388. Ariza, M.E., Cox, B., Martinez, B., Mena-Palomo, I., Zarate, G.J., Williams, M.V ., 2022. Viral dUTPases: Modulators of Innate Immunity. Biomolecules 12 (2). Ayache, S.S., Serratrice, N., Abi Lahoud, G.N., Chalah, M.A., 2022. Fatigue in Multiple Sclerosis: A Review of the Exploratory and Therapeutic Potential of Non-Invasive Brain Stimulation. Front Neurol 13, 813965. Boeschoten, R.E., Braamse, A.M.J., Beekman, A.T.F., Cuijpers, P ., van Oppen, P ., Dekker, J., Uitdehaag, B.M.J., 2017. Prevalence of depression and anxiety in Multiple Sclerosis: A systematic review and meta-analysis. J Neurol Sci 372, 331-341. Chapenko, S., Millers, A., Nora, Z., Logina, I., Kukaine, R.A., Murovska, M., 2003. Correlation between HHV /i2 6 reactivation and multiple sclerosis disease activity. Journal of Medical Virology 69. Comabella, M., Montalban, X., Horga, A., Messmer, B., Kakalacheva, K., Strowig, T., Caballero, E., Münz, C., Lünemann, J.D., 2010. Antiviral immune response in patients with multiple sclerosis and healthy siblings. Multiple Sclerosis Journal 16 (3), 355-358. de Carvalho Jennings Pereira, W.L., Flauzino, T., Alfieri, D.F., Oliveira, S.R., Kallaur, A.P., Simão, A.N.C., Lozovoy, M.A.B., Kaimen-Maciel, D.R., Maes, M., Reiche, E.M.V ., 2020. Prolactin is Not Associated with Disability and Clinical Forms in Patients with Multiple Sclerosis. Neuromolecular Med 22 (1), 73-80. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 23 Debouverie, M., 2009. Gender as a prognostic factor and its impact on the incidence of multiple sclerosis in Lorraine, France. J Neurol Sci 286 (1-2), 14-17. Engdahl, E., Gustafsson, R., Huang, J., Biström, M., Lima Bomfim, I., Stridh, P., Khademi, M., Brenner, N., Butt, J., Michel, A., Jons, D., Hortlund, M., Alonso-Magdalena, L., Hedström, A.K., Flamand, L., Ihira, M., Yoshikawa, T., Andersen, O., Hillert, J., Alfredsson, L., Waterboer, T., Sundström, P., Olsson, T., Kockum, I., Fogdell-Hahn, A., 2019. Increased Serological Response Against Human Herpesvirus 6A Is Associated With Risk for Multiple Sclerosis. Front Immunol 10, 2715. Farrell, R., Antony, D., Wall, G., Clark, D.A., Fisniku, L.K., Swanton, J., Khaleeli, Z., Schmierer, K., Miller, D., Giovannoni, G., 2009. Humoral immune response to EBV in multiple sclerosis is associated with disease activity on MRI. Neurology 73, 32 - 38. Feinstein, A., 2004. The neuropsychiatry of multiple sclerosis. Can J Psychiatry 49 (3), 157-163. Feinstein, A., Magalhaes, S., Richard, J.F., Audet, B., Moore, C., 2014. The link between multiple sclerosis and depression. Nat Rev Neurol 10 (9), 507-517. Fotheringham, J., Jacobson, S., 2005. Human herpesvirus 6 and multiple sclerosis: potential mechanisms for virus-induced disease. Herpes 12 (1), 4-9. Gonsette, R.E., 2008. Neurodegeneration in multiple sclerosis: the role of oxidative stress and excitotoxicity. J Neurol Sci 274 (1-2), 48-53. Hamilton, M., 1959. The assessment of anxiety states by rating. Br J Med Psychol 32 (1), 50-55. Hamilton, M., 1960. A rating scale for depression. J Neurol Neurosurg Psychiatry 23 (1), 56-62. Hanken, K., Sander, C., Schlake, H.P., Kastrup, A., Eling, P., Hildebrandt, H., 2019. Fatigue in Multiple Sclerosis is related to relapses, autonomic dysfunctions and introversion: A quasi- experimental study. Mult Scler Relat Disord 36, 101401. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 24 Jefferies, E., Lambon Ralph, M.A., 2006. Semantic impairment in stroke aphasia versus semantic dementia: a case-series comparison. Brain 129 (Pt 8), 2132-2147. Jongen, P.J., Ter Horst, A.T., Brands, A.M., 2012. Cognitive impairment in multiple sclerosis. Minerva Med 103 (2), 73-96. Kallaur, A.P., Lopes, J., Oliveira, S.R., Simão, A.N., Reiche, E.M., de Almeida, E.R., Morimoto, H.K., de Pereira, W.L., Alfieri, D.F., Borelli, S.D., Kaimen-Maciel, D.R., Maes, M., 2016. Immune-Inflammatory and Oxidative and Nitrosative Stress Biomarkers of Depression Symptoms in Subjects with Multiple Sclerosis: Increased Peripheral Inflammation but Less Acute Neuroinflammation. Mol Neurobiol 53 (8), 5191-5202. Kanchanatawan, B., Sirivichayakul, S., Thika, S., Ruxrungtham, K., Carvalho, A.F., Geffard, M., Anderson, G., Noto, C., Ivanova, R., Maes, M., 2017. Physio-somatic symptoms in schizophrenia: association with depression, anxiety, neurocognitive deficits and the tryptophan catabolite pathway. Metab Brain Dis 32 (4), 1003-1016. Kanchanatawan, B., Sriswasdi, S., Maes, M., 2019. Supervised machine learning to decipher the complex associations between neuro-immune biomarkers and quality of life in schizophrenia. Metab Brain Dis 34 (1), 267-282. Khalesi, Z., Tamrchi, V ., Razizadeh, M.H., Letafati, A., Moradi, P., Habibi, A., Habibi, N., Heidari, J., Noori, M., Nahid Samiei, M., Azarash, Z., Hoseini, M., Saadati, H., Bahavar, A., Farajzade, M., Saeb, S., Hadadi, M., Sorouri Majd, M., Mothlaghzadeh, S., Fazli, P., Asgari, K., Kiani, S.J., Ghorbani, S., 2023. Association between human herpesviruses and multiple sclerosis: A systematic review and meta-analysis. Microbial Pathogenesis 177, 106031. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 25 Khatibi, A., Moradi, N., Rahbari, N., Salehi, T., Dehghani, M., 2020. Development and Validation of Fear of Relapse Scale for Relapsing-Remitting Multiple Sclerosis: Understanding Stressors in Patients. Front Psychiatry 11, 226. Koutsouraki, E., Hatzifilipou, E., Michmizos, D., Cotsavasiloglou, C., Costa, V ., Baloyannis, S., 2011. Increase in interleukin-6 levels is related to depressive phenomena in the acute (relapsing) phase of multiple sclerosis. J Neuropsychiatry Clin Neurosci 23 (4), 442-448. Kurtzke, J.F., 1983. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 33 (11), 1444-1444. Lanz, T.V ., Brewer, R.C., Ho, P .P., Moon, J.S., Jude, K.M., Fernandez, D., Fernandes, R.A., Gomez, A.M., Nadj, G.S., Bartley, C.M., Schubert, R.D., Hawes, I.A., Vazquez, S.E., Iyer, M., Zuchero, J.B., Teegen, B., Dunn, J.E., Lock, C.B., Kipp, L.B., Cotham, V .C., Ueberheide, B.M., Aftab, B.T., Anderson, M.S., DeRisi, J.L., Wilson, M.R., Bashford-Rogers, R.J.M., Platten, M., Garcia, K.C., Steinman, L., Robinson, W.H., 2022. Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature 603 (7900), 321-327. Lundström, W., Gustafsson, R., 2022. Human Herpesvirus 6A Is a Risk Factor for Multiple Sclerosis. Front Immunol 13, 840753. Maes, M., 2023. Major neurocognitive psychosis: a novel schizophrenia endophenotype class that is based on machine learning and resembles Kraepelin's and Bleuler's conceptions. Acta Neuropsychiatr 35 (3), 123-137. Maes, M., Carvalho, A.F., 2018. The Compensatory Immune-Regulatory Reflex System (CIRS) in Depression and Bipolar Disorder. Mol Neurobiol 55 (12), 8885-8903. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 26 Maes, M., De Ruyter, M., Hobin, P ., Suy, E., 1986. The dexamethasone suppression test, the Hamilton Depression Rating Scale and the DSM-III depression categories. J Affect Disord 10 (3), 207-214. Maes, M., Kubera, M., Obuchowiczwa, E., Goehler, L., Brzeszcz, J., 2011. Depression's multiple comorbidities explained by (neuro)inflammatory and oxidative & nitrosative stress pathways. Neuro Endocrinol Lett 32 (1), 7-24. Maes, M., Meltzer, H.Y ., Cosyns, P., Schotte, C., 1994. Evidence for the existence of major depression with and without anxiety features. Psychopathology 27 (1-2), 1-13. Maes, M., Twisk, F.N., Ringel, K., 2012. Inflammatory and cell-mediated immune biomarkers in myalgic encephalomyelitis/chronic fatigue syndrome and depression: inflammatory markers are higher in myalgic encephalomyelitis/chronic fatigue syndrome than in depression. Psychother Psychosom 81 (5), 286-295. McCabe, M.P., 2005. Mood and self-esteem of persons with multiple sclerosis following an exacerbation. J Psychosom Res 59 (3), 161-166. Mohr, D.C., Goodkin, D.E., Islar, J., Hauser, S.L., Genain, C.P ., 2001. Treatment of depression is associated with suppression of nonspecific and antigen-specific T(H)1 responses in multiple sclerosis. Arch Neurol 58 (7), 1081-1086. Moore, P., Hirst, C., Harding, K.E., Clarkson, H., Pickersgill, T.P., Robertson, N.P., 2012. Multiple sclerosis relapses and depression. J Psychosom Res 73 (4), 272-276. Morris, G., Maes, M., 2013a. Myalgic encephalomyelitis/chronic fatigue syndrome and encephalomyelitis disseminata/multiple sclerosis show remarkable levels of similarity in phenomenology and neuroimmune characteristics. BMC Med 11, 205. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 27 Morris, G., Maes, M., 2013b. A neuro-immune model of Myalgic Encephalomyelitis/Chronic fatigue syndrome. Metab Brain Dis 28 (4), 523-540. Morris, G., Reiche, E.M.V ., Murru, A., Carvalho, A.F., Maes, M., Berk, M., Puri, B.K., 2018. Multiple Immune-Inflammatory and Oxidative and Nitrosative Stress Pathways Explain the Frequent Presence of Depression in Multiple Sclerosis. Mol Neurobiol 55 (8), 6282-6306. Nagaraj, K., Taly, A.B., Gupta, A., Prasad, C., Christopher, R., 2013. Prevalence of fatigue in patients with multiple sclerosis and its effect on the quality of life. J Neurosci Rural Pract 4 (3), 278-282. Ormstad, H., Simonsen, C.S., Broch, L., Maes, D.M., Anderson, G., Celius, E.G., 2020. Chronic fatigue and depression due to multiple sclerosis: Immune-inflammatory pathways, tryptophan catabolites and the gut-brain axis as possible shared pathways. Mult Scler Relat Disord 46, 102533. Papiri, G., D'Andreamatteo, G., Cacchiò, G., Alia, S., Silvestrini, M., Paci, C., Luzzi, S., Vignini, A., 2023. Multiple Sclerosis: Inflammatory and Neuroglial Aspects. Curr Issues Mol Biol 45 (2), 1443-1470. Parroche, P., 2011. Modulation of the innate immune response by the oncoviruses EBV and HPV . Université Claude Bernard-Lyon I. Peres, D.S., Rodrigues, P ., Viero, F.T., Frare, J.M., Kudsi, S.Q., Meira, G.M., Trevisan, G., 2022. Prevalence of depression and anxiety in the different clinical forms of multiple sclerosis and associations with disability: A systematic review and meta-analysis. Brain Behav Immun Health 24, 100484. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 28 Polman, C.H., Reingold, S.C., Banwell, B., Clanet, M., Cohen, J.A., Filippi, M., Fujihara, K., Havrdova, E., Hutchinson, M., Kappos, L., 2011. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Annals of neurology 69 (2), 292-302. Roxburgh, R., Seaman, S., Masterman, T., Hensiek, A., Sawcer, S., Vukusic, S., Achiti, I., Confavreux, C., Coustans, M., Le Page, E., 2005. Multiple Sclerosis Severity Score: using disability and disease duration to rate disease severity. Neurology 64 (7), 1144-1151. Šabanagi ć -Hajrić , S., Sulji ć , E., Ku č ukalić , A., 2015. Fatigue during multiple sclerosis relapse and its relationship to depression and neurological disability. Psychiatr Danub 27 (4), 406-412. Siotto, M., Filippi, M.M., Simonelli, I., Landi, D., Ghazaryan, A., V ollaro, S., Ventriglia, M., Pasqualetti, P ., Rongioletti, M.C.A., Squitti, R., Vernieri, F., 2019. Oxidative Stress Related to Iron Metabolism in Relapsing Remitting Multiple Sclerosis Patients With Low Disability. Front Neurosci 13, 86. Skokou, M., Soubasi, E., Gourzis, P., 2012. Depression in multiple sclerosis: a review of assessment and treatment approaches in adult and pediatric populations. ISRN Neurol 2012, 427102. Sommer, P., Kremmer, E., Bier, S., König, S., Zalud, P., Zeppezauer, M., Jones, J.F., Mueller- Lantzsch, N., Grässer, F.A., 1996. Cloning and expression of the Epstein—Barr virus-encoded dUTPase: patients with acute, reactivated or chronic virus infection develop antibodies against the enzyme. Journal of General Virology 77 (11), 2795-2805. Sospedra, M., Martin, R., 2016. Immunology of Multiple Sclerosis. Semin Neurol 36 (2), 115- 127. Staff, N.P., Lucchinetti, C.F., Keegan, B.M., 2009. Multiple Sclerosis With Predominant, Severe Cognitive Impairment. Archives of Neurology 66 (9), 1139-1143. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 29 Sundström, P., Juto, P., Wadell, G., Hallmans, G., Svenningsson, A., Nyström, L., Dillner, J., Forsgren, L., 2004. An altered immune response to Epstein-Barr virus in multiple sclerosis. Neurology 62 (12), 2277-2282. Tiwari, D., Murmu, S., Indari, O., Chandra Jha, H., Kumar, S., 2022. Targeting Epstein /i2 Barr virus dUTPase, an immunomodulatory protein using antiviral, anti /i2 inflammatory, and neuroprotective phytochemicals. Chemistry & Biodiversity 19 (9), e202200527. Trier, N.H., Holm, B.E., Heiden, J., Slot, O., Locht, H., Lindegaard, H., Svendsen, A., Nielsen, C.T., Jacobsen, S., Theander, E., Houen, G., 2018. Antibodies to a strain-specific citrullinated Epstein-Barr virus peptide diagnoses rheumatoid arthritis. Scientific Reports 8 (1), 3684. Vasupanrajit, A., Maes, M., Jirakran, K., Tunvirachaisakul, C., 2024. Complex Intersections Between Adverse Childhood Experiences and Negative Life Events Impact the Phenome of Major Depression. Psychol Res Behav Manag 17, 2161-2178. Villoslada, P., Juste, C., Tintore, M., Llorenç, V ., Codina, G., Pozo-Rosich, P., Montalban, X., 2003. The immune response against herpesvirus is more prominent in the early stages of MS. Neurology 60 (12), 1944-1948. Waldman, W.J., Williams, M.V ., Lemeshow, S., Binkley, P., Guttridge, D., Kiecolt-Glaser, J.K., Knight, D.A., Ladner, K.J., Glaser, R., 2008. Epstein-Barr virus-encoded dUTPase enhances proinflammatory cytokine production by macrophages in contact with endothelial cells: Evidence for depression-induced atherosclerotic risk. Brain, Behavior, and Immunity 22 (2), 215-223. Williams, M.V ., Cox, B., Ariza, M.E., 2016. Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease. Pathogens 6. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 30 Williams, M.V ., Mena-Palomo, I., Cox, B., Ariza, M.E., 2023. EBV dUTPase: A Novel Modulator of Inflammation and the Tumor Microenvironment in EBV-Associated Malignancies, Cancers. Zachrisson, O., Regland, B., Jahreskog, M., Kron, M., Gottfries, C.G., 2002. A rating scale for fibromyalgia and chronic fatigue syndrome (the FibroFatigue scale). Journal of Psychosomatic Research 52 (6), 501-509. Zhang, R., Xu, A., Qin, C., Zhang, Q., Chen, S., Lang, Y ., Wang, M., Li, C., Feng, W., Zhang, R., Jiang, Z., Tang, J., 2017. Pseudorabies Virus dUTPase UL50 Induces Lysosomal Degradation of Type I Interferon Receptor 1 and Antagonizes the Alpha Interferon Response. J Virol 91 (21). . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 31 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 32 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 33 Table 1. Sociodemographic and clinical data in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy controls (HC). Variables Healthy Controls (n=63) RRMS Patients (n=55) F/χ 2 df p-value Age (years) 31.4 (7.1) 29.5 (6.3) 2.18 1/116 0.142 Sex (M/F) 33/30 37/18 2.69 - 0.133 BMI (kg/m2) 26.33 (4.50) 24.94 (3.52) 3.40 1/116 0.068 Education 17.2 (5.1) 18.4 (3.4) 2.04 1/116 0.155 Married/Separated 26/37 31/24 2.67 - 0.139 Smoking (N/Y) 44/19 50/5 8.04 - 0.006 DOI - 5.58 (4.94) 80.31 1/116 <0.0001 MSSS 0 1.71 (1.20) MWU - <0.001 EDSS 0 1.03 (0.11) MWU - <0.001 Total FF score 4.3 (3.5) 23.6 (4.7) 647.51 1/116 <0.001 Total HAMD score 3.1 (2.2) 11.7 (3.5) 262 1/116 <0.0001 Total HAMA score 4.4 (2.9) 13.5 (3.6) 228.93 1/116 <0.0001 PC FFAD (z scores) -0.841 (0.371) 0.964 (0.483) 524.81 1/116 <0.0001 PC DISFADD (z scores) -0.905 (0.129) 1.03 (0.306) 2102.10 1/116 <0.0001 Pure FF symptoms 3.11 (2.86) 17.81 (3.96) 542.37 1/116 <0.0001 Pure HAMD symptoms 0.825 (0.942) 2.40 (1.14) 66.879 1/116 <0.0001 Pure HAMA symptoms 1.63 (1.46) 3.90 (1.65) 62.660 1/116 <0.0001 Pysiosomatic symptoms (z scores) -8.24 (4.30) 9.44 (4.82) 443.401 1/116 <0.0001 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 34 All results are shown as mean (SD). F: results of analysis of variance. χ 2: results of contingency analysis. MWU: Mann-Whitney U test. DOI: duration of illness, MSSS: Multiple Sclerosis Severity Score; EDSS: Expanded Disability Status Scale; FF: Fibro-Fatigue sc ale; HAMD: Hamilton Depression Rating Scale; HAMA: Hamilton Anxiety Rating Scale; PC FFAD: first principal component extracted from FF, HAMD and HA MA scores; PC DISFADD: first PC extracted from EDSS, MSSS, FF, HAMD and HAMA scores. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 35 Table 2: Results of three different principal component analyses (PCA) conducted on disability scales and neuropsychiatric rating scales and immunoglobulin levels against viral antigens, including Epstein-Barr virus (EBV) and Human Herpesvirus-type 6 (HHV-6). Features PCA #1 PCA #2 PCA #3 Total FF 0.954 0.938 0.910 Total HAMD 0.934 0.916 0.842 Total HAMA 0.959 0.924 0.881 MSSS - 0.870 0.877 EDSS - 0.957 0.941 PC EBNA - - 0.800 PC EBV-dUTPases - - 0.861 PC HHV-6-3dUTPases - - 0.881 KMO 0.762 0.856 0.892 %Variance 90.007% 84.883% 76.576% FF: Fibro-Fatigue scale; HAMD: Hamilton Depression Rating Scale; HAMA: Hamilton Anxiety Rating Scale; MSSS: Multiple Sclerosis Severity Score; EDSS: Expanded Disability Status Scale; PC EBNA: first PC extracted from IgG/IgA/IgM responses to Epstein-Barr Virus nuclear antigen 1; PC EBV-dUTPases: first . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 36 PC extracted from IgG/IgA/IgM responses to EBV deoxyuridine-triphosphatase; PC HHV-6-3dUTPases: first principal component extracted from Human Herpes Virus type 6-dUTPase; KMO: Kaiser-Meyer-Olkin metric. Table 3. Intercorrelation matrix between between IgG, IgA and IgM against viral antigens and neuropsychiatric rating scales in relapsing-remitting multiple sclerosis (RRMS). Biomarkers PC DISFADD in all subjects (n=118) PC DISFADD in RRMS (n=55) PC EBNA 0.648** 0.287* PC EBV-dUTPases 0.741** 0.343* PC HHV-6-dUTPases 0.762** 0.326* IgG EBV-dUTPase 0.644** 0.121 IgM EBV-dUTPase 0.634** 0.296* IgA EBV-dUTPase 0.627** 0.306* IgG HHV-6-dUTPase 0.756** 0.042 IgM HHV-6-dUTPase 0.668** 0.222 IgA HHV-6-dUTPase 0.595** 0.401** IgG EBNA 0.490** 0.007 IgM EBNA 0.597** 0.259 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 37 IgA EBNA 0.568** 0.382** *p<0.05, **p<0.01, ***p<0.001. PC EBNA: first PC extracted from IgG/IgA/IgM responses to Epstein-Barr Virus nuclear antigen 1; PC EBV-dUTPases: first PC extracted from IgG/IgA/IgM responses to EBV deoxyuridine-triphosphatase; PC HHV-6-3dUTPases: first principal component extracted from Human Herpes Virus type 6-dUTPase; PC DISFADD: first principal component extracted from disability, fibro-fatigue, depression and anxiety scores. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 38 Table 4. Intercorrelation matrix between IgG, IgA and IgM against viral antigens and neuropsychiatric rating scales in relapsing- remitting multiple sclerosis. Biomarkers PC EBNA PC EBV-dUTPases PC HHV-6-dUTPases Total FF 0.602 ** 0 .662 ** 0 .680 ** Total HAMD 0.502 ** 0 .592 ** 0 .589 ** Total HAMA 0.594 ** 0 .673 ** 0 .658 ** Pure FF 0.591 ** 0 .656 ** 0 .671 ** Pure HAMD 0.331 ** 0 .541 ** 0 .453 ** Pure HAMA 0.423 ** 0 .515 ** 0 .483 ** Pure physiosomatic 0 .607 ** 0 .680 ** 0 .670 ** All p<0.001. PC EBNA: first PC extracted from IgG/IgA/IgM responses to Epstein-Barr Virus nuclear antigen 1; PC EBV-dUTPases: first PC extracted from IgG/IgA/IgM responses to EBV deoxyuridine-triphosphatase; PC HHV-6-3dUTPases: first principal component extracted from Human Herpes Virus type 6-dUTPase; FF: Fibro-Fatigue scale; HAMD: Hamilton Depression Rating Scale; HAMA: Hamilton Anxiety Rating Scale. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 39 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 40 Table 5. Results of multiple regression analysis performed in the total study group with the affective symptoms profiles scores as dependent variables Dependent Variables Explanatory Variables Coefficient statistics Model statistics β t p R2 F df p #1. PC DISFADD Model IgG-HHV-6-dUTPase IgM-HHV-6-dUTPase 0.554 0.326 7.76 4.56 <0.001 <0.001 0.637 100.80 2/115 <0.001 #2. Total FF Model IgG-HHV-6-dUTPase IgM-HHV-6-dUTPase 0.466 0.345 5.77 4.27 <0.001 <0.001 0.535 66.18 2/115 <0.001 #3. Total HAMD Model IgG-HHV-6-dUTPase IgM-HHV-6-dUTPase 0.479 0.214 5.23 2.34 <0.001 0.021 0.402 38.67 2/115 <0.001 #4. Total HAMA Model IgG-HHV-6-dUTPase IgM-HHV-6-dUTPase IgG-EBV-dUTPase 0.221 0.286 0.337 1.99 3.53 3.34 0.049 0.001 0.001 0.536 43.82 3/114 <0.001 #5. Pure FF Model IgG-HHV-6-dUTPase IgM-HHV-6-dUTPase 0.417 0.367 4.96 4.37 <0.001 <0.001 0.498 56.93 2/115 <0.001 #6. Pure HAMD Model IgG-EBV-dUTPase IgM-EBV-dUTPase 0.482 0.182 5.59 2.11 <0.001 0.037 0.383 31.12 2/115 <0.001 #7. Pure HAMA Model IgG-EBV-dUTPase IgM-HHV-6-dUTPase 0.436 0.275 5.16 3.26 <0.001 0.001 0.387 36.22 2/115 <0.001 #8. Pure physiosomatic Model IgG-HHV-6-dUTPase IgM-EBV-dUTPase IgG-EBV-dUTPase 0.316 0.266 0.245 2.86 3.32 2.36 0.005 0.001 0.020 0.510 39.53 3/114 <0.001 FF: Fibro-Fatigue scale; HAMD: Hamilton Depression Rating Scale; HAMA: Hamilton Anxiety Rating Scale; PC DISFADD: first principal component extracted from disability, FF, HAMD and HAMA scores; EBV-dUTPases: Epstein-Barr virus deoxyuridine-triphosphatase; HHV-6: Human Herpes Virus type 6- dUTPase. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 41 Figure 1 Partial regression of the pure physical subdomain of the Fibro-Fatigue (FF) scale on immunoglobulin (Ig)G directed against Human Herpesvirus-6 (HHV/i2 6) deoxyuridine-triphosphatase (dUTPase); p < 0.001 (adjusted for age and IgM reactivity directed to HHV-6-dUTPase). . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 42 Figure 2 . Partial regression of the cognitive subdomain of Hamilton depression rating scale on immunoglobulin (Ig)G directed against Epstein–Barr virus (EBV) deoxyuridine-triphosphatase (dUTPase); p<0.001 (adjusted for IgM-EBV-dUTPase). . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 43 Figure 3. Partial regression of the first principal component extracted from disability, chronic fatigue, depression and anxiety rating scale scores (PC DISFFAD) on immunoglobulin (Ig)A directed against Human Herpesvirus-6 (HHV/i16) deoxyuridine-triphosphatase (dUTPase); p=0.005 (adjusted for age). This regression is performed in RRMS patients only. . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint 44 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 14, 2024. ; https://doi.org/10.1101/2024.10.12.24315393doi: medRxiv preprint