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 (β2AdR) 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-β2AdR 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-β2AdR 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-β2AdR level. Elevated levels of antibodies against β2AdR and M4 receptors in ME/CFS patients support their usage as clinical biomarkers in the diagnostic algorithm of ME/CFS.

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.

Human bocaviruses (HBoVs) 1–4 belong to the Parvoviridae family, and they infect the respiratory or gastrointestinal tracts in children. We investigated the prevalence of HBoV1–4 DNAs in the blood and stool samples, and of HBoV1–4 IgG and IgM in the plasma samples, of children presenting with acute gastroenteritis (AGE). In addition, we identified HBoV co-infections with the five most frequent gastrointestinal pathogens. A total of 83 paired blood and stool samples were collected from children aged five years or less. Infection markers of HBoV1, 2, or 3 (viral DNA in blood and/or stool and/or antibodies) were detected in 61 out of 83 (73.5%) patients. HBoV1, 2, or 3 DNA as a monoinfection was revealed in 18.1%, 2.4%, and 1.2%, respectively, and 21.7% in total. In 56.1% of the HBoV DNA-positive patients, the presence in stool of another virus—most frequently norovirus or rotavirus—was observed. In conclusion, this study, for the first time, illustrates the prevalence and genetic diversity of HBoVs in Latvian children with gastroenteritis, and shows a widespread distribution of these viruses in the community. HBoV1 and 2 are commonly found as single infectious agents in children with AGE, suggesting that the viruses can be as pathogenic by themselves as other enteric agents are.

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.

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0–6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.