Reactivation of herpesvirus type 6 and IgA/IgM-mediated responses to activin-A underpin long COVID, including affective symptoms and chronic fatigue syndrome.

BACKGROUND: Persistent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reactivation of dormant viruses, and immune-oxidative responses are involved in long COVID. OBJECTIVES: To investigate whether long COVID and depressive, anxiety, and chronic fatigue syndrome (CFS) symptoms are associated with IgA/IgM/IgG to SARS-CoV-2, human herpesvirus type 6 (HHV-6), Epstein-Barr Virus (EBV), and immune-oxidative biomarkers. METHODS: We examined 90 long COVID patients and ninety healthy controls. We measured serum IgA/IgM/IgG against HHV-6 and EBV and their deoxyuridine 5'-triphosphate nucleotidohydrolase (duTPase), SARS-CoV-2, and activin-A, C-reactive protein (CRP), advanced oxidation protein products (AOPP), and insulin resistance (HOMA2-IR). RESULTS: Long COVID patients showed significant elevations in IgG/IgM-SARS-CoV-2, IgG/IgM-HHV-6, and HHV-6-duTPase, IgA/IgM-activin-A, CRP, AOPP, and HOMA2-IR. Neural network analysis yielded a highly significant predictive accuracy of 80.6% for the long COVID diagnosis (sensitivity: 78.9%, specificity: 81.8%, area under the ROC curve = 0.876); the topmost predictors were as follows: IGA-activin-A, IgG-HHV-6, IgM-HHV-6-duTPase, IgG-SARS-CoV-2, and IgM-HHV-6 (all positively) and a factor extracted from all IgA levels to all viral antigens (inversely). The top 5 predictors of affective symptoms due to long COVID were IgM-HHV-6-duTPase, IgG-HHV-6, CRP, education, IgA-activin-A (predictive accuracy of r = 0.636). The top 5 predictors of CFS due to long COVID were in descending order: CRP, IgG-HHV-6-duTPase, IgM-activin-A, IgM-SARS-CoV-2, and IgA-activin-A (predictive accuracy: r = 0.709). CONCLUSION: Reactivation of HHV-6, SARS-CoV-2 persistence, and autoimmune reactions to activin-A combined with activated immune-oxidative pathways play a major role in the pathophysiology of long COVID as well as the severity of its affective symptoms and CFS.

Human Herpesvirus-6 Reactivation, Mitochondrial Fragmentation, and the Coordination of Antiviral and Metabolic Phenotypes in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multifactorial disorder with many possible triggers. Human herpesvirus (HHV)-6 and HHV-7 are two infectious triggers for which evidence has been growing. To understand possible causative role of HHV-6 in ME/CFS, metabolic and antiviral phenotypes of U2-OS cells were studied with and without chromosomally integrated HHV-6 and with or without virus reactivation using the histone deacetylase inhibitor trichostatin-A. Proteomic analysis was conducted by pulsed stable isotope labeling by amino acids in cell culture analysis. Antiviral properties that were induced by HHV-6 transactivation were studied in virus-naive A549 cells challenged by infection with influenza-A (H1N1) or HSV-1. Mitochondria were fragmented and 1-carbon metabolism, dUTPase, and thymidylate synthase were strongly induced by HHV-6 reactivation, whereas superoxide dismutase 2 and proteins required for mitochondrial oxidation of fatty acid, amino acid, and glucose metabolism, including pyruvate dehydrogenase, were strongly inhibited. Adoptive transfer of U2-OS cell supernatants after reactivation of HHV-6A led to an antiviral state in A549 cells that prevented superinfection with influenza-A and HSV-1. Adoptive transfer of serum from 10 patients with ME/CFS produced a similar fragmentation of mitochondria and the associated antiviral state in the A549 cell assay. In conclusion, HHV-6 reactivation in ME/CFS patients activates a multisystem, proinflammatory, cell danger response that protects against certain RNA and DNA virus infections but comes at the cost of mitochondrial fragmentation and severely compromised energy metabolism.

Serum levels of matrix metalloproteinase-2, -9, and vitamin D in patients with multiple sclerosis with or without herpesvirus-6 seropositivity.

In recent years, extreme attention has been focused on the role of human herpesvirus-6 (HHV-6) in multiple sclerosis (MS) pathogenesis. However, the pathogenesis of MS associated with HHV-6 infection remains unknown. In this study, we measured the serum levels of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and vitamin D levels in MS patients with HHV-6 infection and MS patients without HHV-6 infection. Five hundred sixty (including 300 females and 260 males) MS patients along with 560 healthy subjects were analyzed for HHV-6 seropositivity using enzyme-linked immunosorbent assay (ELISA). Subsequently, we measured the serum levels of MMP-2, MMP-9, and vitamin D levels in MS patients with HHV-6 infection and MS patients without HHV-6 infection by ELISA. About 90.7% of MS patients (508/560) were seropositive for HHV-6, while 82.3% (461/560) of healthy subjects were seropositive for this virus (p = 0.001). Moreover, there was a significant increase in the levels of MMP-2, MMP-9, and lower vitamin D in the serum samples of MS patients when compared with healthy subjects. Additionally, we demonstrated that the MMP-9 levels in seropositive MS patients were significantly higher than seronegative MS patients (p =  0.001). Finally, our results demonstrated that the mean of expanded disability status scale (EDSS) in seropositive MS patients was significantly higher in comparison to seronegative MS patients (p < 0.05). In conclusion, we suggest that the HHV-6 infection may play a role in MS pathogenesis.

Serum levels of matrix metalloproteinase-2, -9, and vitamin D in patients with multiple sclerosis with or without herpesvirus-6 seropositivity

ABSTRACT In recent years, extreme attention has been focused on the role of human herpesvirus-6 (HHV-6) in multiple sclerosis (MS) pathogenesis. However, the pathogenesis of MS associated with HHV-6 infection remains unknown. In this study, we measured the serum levels of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and vitamin D levels in MS patients with HHV-6 infection and MS patients without HHV-6 infection. Five hundred sixty (including 300 females and 260 males) MS patients along with 560 healthy subjects were analyzed for HHV-6 seropositivity using enzyme-linked immunosorbent assay (ELISA). Subsequently, we measured the serum levels of MMP-2, MMP-9, and vitamin D levels in MS patients with HHV-6 infection and MS patients without HHV-6 infection by ELISA. About 90.7% of MS patients (508/560) were seropositive for HHV-6, while 82.3% (461/560) of healthy subjects were seropositive for this virus (p = 0.001). Moreover, there was a significant increase in the levels of MMP-2, MMP-9, and lower vitamin D in the serum samples of MS patients when compared with healthy subjects. Additionally, we demonstrated that the MMP-9 levels in seropositive MS patients were significantly higher than seronegative MS patients (p = 0.001). Finally, our results demonstrated that the mean of expanded disability status scale (EDSS) in seropositive MS patients was significantly higher in comparison to seronegative MS patients (p < 0.05). In conclusion, we suggest that the HHV-6 infection may play a role in MS pathogenesis.

Correlations of cytokine levels in cerebrospinal fluid and peripheral blood with outcome of HHV-6B encephalitis after hematopoietic stem cell transplantation.

BACKGROUND: Human herpesvirus (HHV)-6B encephalitis has been recognized as a serious complication after allogeneic hematopoietic cell transplantation (allo-HCT). Little is known about the pathogenic mechanism for its progression. STUDY DESIGN: We retrospectively evaluated the 16 kinds of cytokines and chemokines in cerebrospinal fluid (CSF) and plasma in patients who developed HHV-6B encephalitis. Among a total of 20 patients, 12 were categorized as the poor prognosis group (died of encephalitis; n = 8 and retained sequelae; n = 4), and other eight patients were categorized as the good prognosis group (complete recovery; n = 8). RESULTS: Concentrations of CSF IL-6 and IL-8 at the onset of encephalitis were significantly higher in the poor prognosis group than in the good prognosis group (median CSF IL-6, 28.27 pg/mL vs 14.32 pg/mL, P = .004; median CSF IL-8, 128.70 pg/mL vs 59.43 pg/mL, P = .043). Regarding plasma, the concentration of each cytokine at the onset of encephalitis was not significantly different between the two groups, except IL-5. However, higher levels of IL-6, IL-7, and MCP-1 and lower levels of IL-12 were observed 1 week before the development of encephalitis in patients with poor prognosis (median IL-6; 464.17 pg/mL vs 47.82 pg/mL, P = .02; median IL-12; 1.63 pg/mL vs 6.57 pg/mL, P = .03). CONCLUSION: We found that one week before onset of HHV-6B encephalitis, poor prognosis patients had high plasma concentrations of IL-6, IL-7, and MCP-1 and low concentrations of IL-12. At the onset of encephalitis, high concentrations of IL-6 and IL-8 in CSF were more common in the poor prognosis group, consistent with other evidence that IL-6 can have a role in CNS disturbances. Our findings show that specific cytokine status is associated with severe brain damage in patients with HHV-6B encephalitis, demonstrate prognostic value of plasma IL-6 concentrations, and suggest evaluation of anti-cytokine therapeutics in patients with HHV-6B encephalitis.

Primary infection with human herpes virus type 6, post-pediatric liver transplantation-A pathogen to remember.

BACKGROUND: In recent years, liver transplantation (LT) has become a well-accepted therapeutic modality for children with end-stage liver disease, with transplantation surgery being performed at a younger age. Human herpes virus 6 (HHV-6) infection occurs in most children within the first 2 years of life, therefore, data on primary HHV-6 infection in pediatric liver transplant recipients is scarce. OBJECTIVE: To describe the course of primary HHV-6 infection after pediatric LT. METHODS: Medical files, between the years 2015-2016, of post-LT pediatric patients with suspected primary HHV-6 infection were reviewed. Clinical and laboratory data for enrolled cases were evaluated. Primary infection was defined as DNAemia in children who were seronegative prior to transplantation or seroconversion from negative to positive IgG posttransplantation. RESULTS: Four cases of primary HHV-6 (type B) infection were identified among the 26 children who had undergone LT at our center during the study period. All patients were <1 year old and presented with fever, hepatitis, and elevated inflammatory markers, most (75%) within a short-period posttransplantation. All were initially treated with empiric antibiotics for a suspected bacterial infection and three underwent liver biopsy, one showing signs of rejection. Three were treated with antiviral therapy with a gradual resolution of symptoms. DISCUSSION: Primary HHV-6 should be taken into account in young children shortly after LT, especially when presenting with fever and elevated liver enzymes. Treatment with antiviral therapy should be considered. CONCLUSIONS: In young infants post-LT, a high index of suspicion may promote early detection of HHV-6 primary infection and prevent serious complications.

Comparison of HHV-6 DNA detection in plasma and whole blood in allogeneic hematopoietic stem cell transplant recipients: frequent false-positive results for active HHV-6 infection using whole blood samples.

In this prospective observational study, we compared the human herpesvirus-6 (HHV-6) DNA load in serially collected paired plasma and whole blood (WB) samples from allogeneic hematopoietic stem cell transplantation (HSCT) recipients. A total of 721 paired samples were collected from 68 recipients. The positive rate for HHV-6 DNA was 9.7 and 35.0% in plasma and WB samples, respectively (P < 0.001). The correlation of HHV-6 DNA load between plasma and WB was poor (R2 = 0.250). After reaching peak levels, HHV-6 DNA showed a delayed decrease in WB in comparison with plasma (median, 28 versus 7 days, P < 0.001). We additionally tested HHV-6 mRNA status in 95 samples from eight patients. To identify positive HHV-6 mRNA, plasma HHV-6 DNA showed 55.0% sensitivity and 100% specificity, whereas WB HHV-6 DNA showed 90.0% sensitivity and 68.0% specificity. The false-positive rate for identifying positive HHV-6 mRNA was 0% for plasma HHV-6 DNA and 32.0% for WB HHV-6 DNA. Although WB was more sensitive than plasma for detecting HHV-6 reactivation, the rates of false positivity for active HHV-6 infection were higher for WB than for plasma.

Kinetics of Double-Stranded DNA Viremia After Allogeneic Hematopoietic Cell Transplantation.

Background: Improved understanding of double-stranded DNA (dsDNA) virus kinetics after hematopoietic cell transplantation (HCT) would facilitate development of therapeutic strategies. Methods: We tested weekly plasma samples from 404 patients through day 100 after allogeneic HCT for cytomegalovirus (CMV), human herpesvirus (HHV) 6A and 6B, BK polyomavirus (BKV), adenovirus (AdV), and Epstein-Barr virus (EBV) using quantitative polymerase chain reaction. Episodes lasting ≤1 week were defined as blips and >1 week as persistent. We described virus-specific kinetics, analyzed the association of virus area under the curve (AUC) with overall mortality, and identified risk factors for persistent episodes. Results: We identified 428 episodes of CMV, 292 of BKV, 224 of HHV-6B, 46 of AdV, and 53 of EBV. CMV and BKV had the highest proportions of persistent episodes (68% and 80%, respectively). Detection and kinetics varied by virus. HHV-6B episodes reached maximum levels fastest and had the shortest intervals between detection and end-organ disease. End-organ disease occurred within 14 days of viremia in 68% of cases, generally during persistent episodes. For all viruses, higher viral load AUC increased risk for overall mortality through day 365, persistent episodes had higher viral load than blips, and higher first positive viral load significantly increased risk for persistent episodes. First viral load >2 log10 copies/mL (range, 2.04-3.06 per virus) had high specificity for persistent episodes. Conclusions: Persistent high viral load dsDNA viremia episodes after allogeneic HCT predict mortality. Virus-specific kinetics can guide timing and thresholds for early intervention in studies of novel agents.

Transmission of chromosomally integrated human herpesvirus 6 via cord blood transplantation.

Chromosomally integrated human herpesvirus 6 (ciHHV-6) can be transmitted via allogeneic hematopoietic cell transplantation. To date, only a few cases have been reported. Here, we report a case identified as transmission of ciHHV-6 via cord blood transplantation. Distinguishing transmission of ciHHV-6 from HHV-6 reactivation in cases with high titer of HHV-6 DNA load after transplantation is important to prevent unnecessary exposure to antiviral drugs that could be toxic.

Increase in Peripheral CD3-CD56brightCD16- Natural Killer Cells in Hashimoto's Thyroiditis Associated with HHV-6 Infection.

Hashimoto's thyroiditis (HT) is a very common autoimmune disease of the thyroid. In addition to genetic background, several viruses, including herpesviruses, have been suggested to play a role as possible environmental triggers of disease, but conclusive data are still lacking. Previous results showed that HT patients have an increased cellular immune response directed against the HHV-6 U94 protein and increased NK activity directed against HHV-6 infected thyrocytes.In this study, we characterized the antiviral antibody response and the NK cells activity and subtype in HHV-6 infected HT patients. The results showed that HT subjects have increased prevalence and titer of anti-U94 antibodies and a higher amount of CD3-CD56(bright)CD16(-)NK cell percentages compared to controls. Furthermore, the cell activation of CD3(-)CD56(bright) NK cells in HT patients significantly correlates with TPO and Tg Ab levels.The results suggest that HHV-6 might contribute to HT development, increasing NK cell secretion of inflammatory cytokines that could sustain the persistence of an inflammatory status in HT patients.

Human herpesvirus-6 infection-associated acute encephalopathy without skin rash.

BACKGROUND: Human herpesvirus-6 (HHV-6) is the etiological agent of exanthema subitum-associated encephalopathy, which usually occurs in children younger than 3 years. Brain imaging shows various abnormalities. PATIENT: A previously healthy 4-year-old girl developed acute encephalopathy with clinical features consisting of fever, repetitive seizures, and a disturbance of consciousness. The patient did not show skin rash suggestive of exanthema subitum during the course of her illness. The primary HHV-6 infection was diagnosed based on the absence of IgG against HHV-6 and identification of the virus DNA in the acute phase serum and a significant increase of the anti-HHV-6 IgG titers in the convalescent phase sera. Diffusion-weighted images showed transient high signal intensity in the bilateral periventricular white matter and splenium of the corpus callosum and in the gray matter structures such as the bilateral basal ganglia and thalami. Upon therapy with steroid and γ-globulin, the patient recovered without any neurological deficits. CONCLUSION: Primary HHV-6 infection can cause acute encephalopathy without exanthema subitum. The etiological diagnosis is possible only by examining the blood and cerebrospinal fluid, when the patient shows no skin rash. This condition should be included in the differential diagnosis of acute encephalopathy even in patients older than 3 years.

Seroprevalence of HHV-6 and HHV-8 among blood donors in Greece.

BACKGROUND: Herpes viruses infection transmitted through healthy but infected blood donors pose a danger to herpes-naive immunocompromised recipients. The risk of transfusion-related HHV-8 transmission is different in endemic and not endemic areas. HHV-6 and HHV-8 seroprevalence and viral load among blood donors have been reported from different countries. The aim of our study was to assess the seroprevalence of HHV-8 and HHV-6 in volunteer blood donors from Greece which is unknown. FINDINGS: Serum samples from 179 healthy blood donors were tested for the presence of IgG antibodies against HHV-6 and HHV-8 with ELISA. None of the 179 donors of Greek origin tested was positive for HHV-8. HHV-6 seropositivity was assessed in 160 blood donors' samples and was found to be 78.75% (126/160). The HHV-6 seroprevalence did not differ either between males and females or among different decade age groups. CONCLUSIONS: The fact, that no blood donor was positive for HHV-8 IgG antibodies indicates that the risk for transfusion related HHV-8 transmission in Greece, if any, is negligible and does not warrant broad testing for HHV-8. Definitely further studies are needed, in order to clarify the potential risk of HHV-6 transmission.

Reactivation of multiple viruses in patients with sepsis.

A current controversy is whether patients with sepsis progress to an immunosuppressed state. We hypothesized that reactivation of latent viruses occurred with prolonged sepsis thereby providing evidence of clinically-relevant immunosuppression and potentially providing a means to serially-monitor patients' immune status. Secondly, if viral loads are markedly elevated, they may contribute to morbidity and mortality. This study determined if reactivation of herpesviruses, polyomaviruses, and the anellovirus TTV occurred in sepsis and correlated with severity. Serial whole blood and plasma samples from 560 critically-ill septic, 161 critically-ill non-septic, and 164 healthy age-matched patients were analyzed by quantitative-polymerase-chain-reaction for cytomegalovirus (CMV), Epstein-Barr (EBV), herpes-simplex (HSV), human herpes virus-6 (HHV-6), and TTV. Polyomaviruses BK and JC were quantitated in urine. Detectable virus was analyzed with respect to secondary fungal and opportunistic bacterial infections, ICU duration, severity of illness, and survival. Patients with protracted sepsis had markedly increased frequency of detectable virus. Cumulative viral DNA detection rates in blood were: CMV (24.2%), EBV (53.2%), HSV (14.1%), HHV-6 (10.4%), and TTV (77.5%). 42.7% of septic patients had presence of two or more viruses. The 50% detection rate for herpesviruses was 5-8 days after sepsis onset. A small subgroup of septic patients had markedly elevated viral loads (>104-106 DNA copies/ml blood) for CMV, EBV, and HSV. Excluding TTV, DNAemia was uncommon in critically-ill non-septic patients and in age-matched healthy controls. Compared to septic patients without DNAemia, septic patients with viremia had increased fungal and opportunistic bacterial infections. Patients with detectable CMV in plasma had higher 90-day mortality compared to CMV-negative patients; p<0.05. Reactivation of latent viruses is common with prolonged sepsis, with frequencies similar to those occurring in transplant patients on immunosuppressive therapy and consistent with development of an immunosuppressive state. Whether reactivated latent viruses contribute to morbidity and mortality in sepsis remains unknown.

Detection of HHV-6-specific mRNA and antigens in PBMCs of individuals with chromosomally integrated HHV-6 (ciHHV-6).

After inheritance of chromosomally integrated HHV-6 (ciHHV-6), viral DNA is found in every nucleated cell. The prevalence of ciHHV-6 is estimated to be 0.2-5% of humans. There are conflicting data on the potential for replication, possibly leading to clinical implications. We analysed peripheral blood mononuclear cells (PBMCs) from individuals with ciHHV-6 proven by fluorescence in situ hybridization (FISH) for HHV-6-specific mRNA (U94, U42, U22) and antigens by means of reverse transcription PCR and an indirect immunoperoxidase staining. U94 transcripts indicative of latent infection were detected in six (54.5%) out of 11 individuals at least once. Transcripts indicative of lytic infection (i.e. U42 and U22) were detected in four (36.4%) out of 11 individuals at least once. HHV-6 antigen was detected in seven (70%) out of 10 individuals at least once. The presence of viral mRNA and proteins supports virus gene expression from ciHHV-6, which may lead to virus replication. Considering the properties of active HHV-6 infection together with obvious replicative activity in individuals with ciHHV-6, pathophysiological effects leading to clinical consequences of chromosomally integrated viral DNA might be considered.