Reactivation of a transplant recipient's inherited human herpesvirus 6 and implications to the graft.

BACKGROUND: The implications of inherited chromosomally integrated human herpesvirus 6 (iciHHV-6) in solid organ transplantation remain uncertain. Although this trait has been linked to unfavorable clinical outcomes, an association between viral reactivation and complications has only been conclusively established in a few cases. In contrast to these studies, which followed donor-derived transmission, our investigation is the first to examine the pathogenicity of a recipient´s iciHHV-6B and its impact on the graft. METHODS: We used hybrid capture sequencing for in-depth analysis of the viral sequences reconstructed from sequential liver biopsies. Moreover, we investigated viral replication through in situ hybridization (U38-U94 genes), real-time PCR (U89/U90 genes), immunohistochemistry, and immunofluorescence (against viral lysate). We also performed whole transcriptome sequencing of the liver biopsies to profile the host immune response. RESULTS: We report a case of reactivation of a recipient´s iciHHV-6B and subsequent infection of the graft. Using a novel approach integrating the analysis of viral and mitochondrial DNAs, we located the iciHHV-6B intra-graft. We demonstrated active replication via the emergence of viral minor variants across time points, in addition to positive viral mRNAs and antigen stainings in tissue sections. Furthermore, we detected significant upregulation of cell surface molecules, transcription factors, and cytokines associated with antiviral immune responses, arguing against immunotolerance. CONCLUSIONS: Our analysis underscores the potential pathological impact of iciHHV-6B, emphasizing the need for close monitoring of reactivation in transplant recipients. Most crucially, it highlights the critical role that the host's virome can play in shaping the outcome of transplantation, urging further investigations.

Circulating miRNAs Expression in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multifactorial disease that causes increasing morbidity worldwide, and many individuals with ME/CFS symptoms remain undiagnosed due to the lack of diagnostic biomarkers. Its etiology is still unknown, but increasing evidence supports a role of herpesviruses (including HHV-6A and HHV-6B) as potential triggers. Interestingly, the infection by these viruses has been reported to impact the expression of microRNAs (miRNAs), short non-coding RNA sequences which have been suggested to be epigenetic factors modulating ME/CFS pathogenic mechanisms. Notably, the presence of circulating miRNAs in plasma has raised the possibility to use them as valuable biomarkers for distinguishing ME/CFS patients from healthy controls. Thus, this study aimed at determining the role of eight miRNAs, which were selected for their previous association with ME/CFS, as potential circulating biomarkers of the disease. Their presence was quantitatively evaluated in plasma from 40 ME/CFS patients and 20 healthy controls by specific Taqman assays, and the results showed that six out of the eight of the selected miRNAs were differently expressed in patients compared to controls; more specifically, five miRNAs were significantly upregulated (miR-127-3p, miR-142-5p, miR-143-3p, miR-150-5p, and miR-448), and one was downmodulated (miR-140-5p). MiRNA levels directly correlated with disease severity, whereas no significant correlations were observed with the plasma levels of seven pro-inflammatory cytokines or with the presence/load of HHV-6A/6B genome, as judged by specific PCR amplification. The results may open the way for further validation of miRNAs as new potential biomarkers in ME/CFS and increase the knowledge of the complex pathways involved in the ME/CFS development.

The persistent viral infections in the development and severity of myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multifactorial disease with an unexplained aetiology in which viral infections are possible trigger factors. The aim of this study was to determine the involvement of human herpesvirus (HHV)-6A/B, HHV-7, and parvovirus B19 (B19V) in the etiopathogenesis of ME/CFS. METHODS: 200 patients with clinically diagnosed ME/CFS and 150 apparently healthy individuals were enrolled in this study. Single-round, nested, and quantitative real-time polymerase chain reactions (PCR) were used to detect the presence and load of HHV-6A/B, HHV-7, and B19V. HHV-6A and HHV-6B were distinguished by PCR and restriction analysis. Immunoenzymatic assays were applied to estimate the presence of virus-specific antibodies and the level of cytokines. RESULTS: HHV-6A/B, HHV-7, and B19V specific antibodies were detected among patients and healthy individuals in 92.1% and 76.7%, 84.6% and 93.8%, and 78% and 67.4% of cases. HHV-6B had 99% of HHV-6 positive patients. Latent HHV-6A/B, HHV-7, and B19V infection/co-infection was observed in 51.5% of the patients and 76.7% of the healthy individuals, whereas active-45% of the ME/CFS patients and 8.7% of healthy individuals. HHV-6A/B load in patients with a persistent infection/co-infection in a latent and active phase was 262 and 653.2 copies/106 cells, whereas HHV-7 load was 166.5 and 248.5 copies/106 cells, and B19V-96.8 and 250.8 copies/106 cells, respectively. ME/CFS patients with persistent infection in an active phase had a higher level of pro-inflammatory cytokines (interleukin(IL)-6, tumor necrosis factor-alpha(TNF-α) and IL-12) and anti-inflammatory (IL-10) than with a persistent infection in a latent phase. A significant difference was revealed in the levels of TNF-α, IL-12, and IL-10 among the patient groups without infection, with latent infection/co-infection, active single, double and triple co-infection. The levels of TNF-α, IL-12, and IL-10 are significantly higher in patients with severe compared with a moderate course of ME/CFS. CONCLUSIONS: Significantly more persistent HHV-6A/B, HHV-7, and B19V infection/co-infection in an active phase with a higher viral load and elevated levels of pro- and anti-inflammatory cytokines among patients with ME/CFS than healthy individuals indicate the importance of these infections/co-infections in ME/CFS development. The presence of these infections/co-infections influences the ME/CFS clinical course severity.

Biomarkers in the diagnostic algorithm of myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disease that is mainly diagnosed based on its clinical symptoms. Biomarkers that could facilitate the diagnosis of ME/CFS are not yet available; therefore, reliable and clinically useful disease indicators are of high importance. The aim of this work was to analyze the association between ME/CFS clinical course severity, presence of HHV-6A/B infection markers, and plasma levels of autoantibodies against adrenergic and muscarinic acetylcholine receptors. A total of 134 patients with ME/CFS and 33 healthy controls were analyzed for the presence of HHV-6A/B using PCRs, and antibodies against beta2-adrenergic receptors (ß2AdR) and muscarinic acetylcholine receptors (M3 AChR and M4 AChR) using ELISAs. HHV-6A/B U3 genomic sequence in whole-blood DNA was detected in 19/31 patients with severe ME/CFS, in 18/73 moderate ME/CFS cases, and in 7/30 mild ME/CFS cases. Severity-related differences were found among those with a virus load of more than 1,000 copies/106 PBMCs. Although no disease severity-related differences in anti-ß2AdR levels were observed in ME/CFS patients, the median concentration of these antibodies in plasma samples of ME/CFS patients was 1.4 ng/ml, while in healthy controls, it was 0.81 ng/ml, with a statistically significant increased level in those with ME/CFS (p = 0.0103). A significant difference of antibodies against M4 AChR median concentration was found between ME/CFS patients (8.15 ng/ml) and healthy controls (6.45 ng/ml) (p = 0.0250). The levels of anti-M4 plotted against disease severity did not show any difference; however, increased viral load correlates with the increase in anti-M4 level. ME/CFS patients with high HHV-6 load have a more severe course of the disease, thus confirming that the severity of the disease depends on the viral load-the course of the disease is more severe with a higher viral load. An increase in anti-M4 AchR and anti-ß2AdR levels is detected in all ME/CFS patient groups in comparison to the control group not depending on ME/CFS clinical course severity. However, the increase in HHV-6 load correlates with the increase in anti-M4 level, and the increase in anti-M4 level, in turn, is associated with the increase in anti-ß2AdR level. Elevated levels of antibodies against ß2AdR and M4 receptors in ME/CFS patients support their usage as clinical biomarkers in the diagnostic algorithm of ME/CFS.

Human Parvoviruses May Affect the Development and Clinical Course of Meningitis and Meningoencephalitis.

Meningitis and meningoencephalitis are neurological inflammatory diseases, and although routine diagnostics include testing of a wide range of pathogens, still in many cases, no causative agent is detected. Human parvovirus B19 (B19V), human bocaviruses 1-4 (HBoV1-4), and human parvovirus 4 (hPARV4) are members of the Parvoviridae family and are associated with a wide range of clinical manifestations including neurological disorders. The main aim of this study was to determine whether human parvoviruses infection markers are present among patients with meningitis/meningoencephalitis in Latvia as well as to clarify the role of these viruses on the clinical course of the mentioned diseases. Our study revealed HBoV1-4 and B19V genomic sequences in 52.38% and 16.67% of patients, respectively. Furthermore, symptoms such as the presence of a headache and its severity, fatigue, disorientation, and difficulties to concentrate were significantly frequently present in patients with active parvovirus infection in comparison with parvoviruses negative patients, therefore we suggest that HBoV1-4 and B19V infection should be included in the diagnostics to reduce the number of meningitis/meningoencephalitis with unknown/unexplained etiology.

Persistent Roseoloviruses Infection in Adult Patients with Epilepsy.

BACKGROUND: Human herpesviruses (HHV)-6A, HHV-6B and HHV-7 are considered to be involved in the pathogenesis of epilepsy, a common neurological disorder. The objective of this study was to determine the association of roseoloviruses infection with epilepsy. METHODS: 53 epilepsy patients and 104 ordinary blood donors were analyzed to determine presence of virus-specific antibodies by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), genomic sequences, viral load and gene expression by polymerase chain reactions (PCRs) and restriction analysis, HHV-6 protein expression by IFA and level of cytokines by ELISA. RESULTS: Roseoloviruses genomic sequences in DNA samples from whole blood were found in 86.8% of patients versus 54.8% of controls and active infection was revealed only in patients with epilepsy (19.6% of roseolovirus-positive patients). Significantly higher viral load and more frequent gene expression was detected in patients compared to the controls. HHV-6-encoded protein expression was demonstrated in 53.3% of patients with previously detected HHV-6 DNA. Changes in level of cytokines were determined in patients with elevated viral load compared to the patients without elevated viral loads and to the controls. CONCLUSIONS: Results on frequent active HHV-6 and HHV-7 infection in epilepsy patient' peripheral blood indicate on possible involvement of these viruses in the disease development.

Genetic Diversity and Phylogenetic Analysis of Human Bocavirus 2 in Pediatric Patients with Acute Gastroenteritis in Taiwan.

Human bocavirus (HBoV) is a causative agent of respiratory and gastrointestinal diseases worldwide. Four HBoV species (HBoV1-4) have been identified so far. Although a previous report has documented the HBoV association with acute gastroenteritis (AGE) in Taiwan, their epidemiology, genetic diversity, and phylogenetic relationships remain unclear. In this study, we focused on an investigation of these unsolved issues, which will help to reveal molecular epidemiology and phylogeny of the circulating HBoV2 in Taiwan. A total of 176 stool samples were collected from children with AGE for this study. PCR amplification and sequencing on the VP1 gene region were used to identify species. Phylogenetic analysis was conducted by maximum-likelihood and neighbor-joining methods. Selection pressure was also estimated to obtain HBoV evolutionary information. Our results showed the prevalence of HBoV in AGE children was 8.5%, of which HBoV1 was the predominant species (6.3%), followed by HBoV2 (2.3%). Phylogenetic analysis showed those Taiwanese HBoV2 strains have significant genetic variability and can be divided into two clusters. One belongs to HBoV2 genotype A and the other forms an independent unclassified cluster. The nucleotide distance between that independent cluster and the known HBoV2 genotypes was more than 5%, suggesting a new HBoV2 genotype. No positive selection site was found and the virus was under purifying selection. This is the first report to reveal HBoV2 genetic diversity and phylogenetic relationships among AGE children in Taiwan. We find that HBoV2 may have been introduced into the country by multiple origins, and a potential new HBoV2 genotype is proposed.

Acute human bocavirus 1 infection in child with life-threatening bilateral bronchiolitis and right-sided pneumonia: a case report.

BACKGROUND: Human bocavirus 1 is a commonly detected human parvovirus. Many studies have shown human bocavirus 1 as a pathogen in association with acute respiratory tract infections in children. However, because human bocavirus 1 persists in the upper airways for extensive time periods after acute infection, the definition and diagnostics of acute human bocavirus 1 infection is challenging. Until now, detection of human bocavirus 1 exclusively, high viral load in respiratory samples, and viremia have been associated with a clinical picture of acute respiratory illness. There are no studies showing detection of human bocavirus 1 messenger ribonucleic acid in the peripheral blood mononuclear cells as a diagnostic marker for acute lower respiratory tract infection. CASE PRESENTATION: We report the case of a 17-month-old Latvian boy who presented in intensive care unit with acute bilateral bronchiolitis, with a history of rhinorrhea and cough for 6 days and fever for the last 2 days prior to admission, followed by severe respiratory distress and tracheal intubation. Human bocavirus 1 was the only respiratory virus detected by a qualitative multiplex polymerase chain reaction panel. For the diagnosis of acute human bocavirus 1 infection, both molecular and serological approaches were used. Human bocavirus 1 deoxyribonucleic acid (DNA) was detected simultaneously in nasopharyngeal aspirate, stool, and blood, as well as in the corresponding cell-free blood plasma by qualitative and quantitative polymerase chain reaction, revealing high DNA-copy numbers in nasopharyngeal aspirate and stool. Despite a low-load viremia, human bocavirus 1 messenger ribonucleic acid was found in the peripheral blood mononuclear cells. For detection of human bocavirus 1-specific antibodies, non-competitive immunoglobulin M and competitive immunoglobulin G enzyme immunoassays were used. The plasma was positive for both human bocavirus 1-specific immunoglobulin M and immunoglobulin G antibodies. CONCLUSIONS: The presence of human bocavirus 1 genomic DNA in blood plasma and human bocavirus 1 messenger ribonucleic acid in peripheral blood mononuclear cells together with human bocavirus 1-specific immunoglobulin M are markers of acute human bocavirus 1 infection that may cause life-threatening acute bronchiolitis.