Better detection of Torque teno virus in children with leukemia by metagenomic sequencing than by quantitative PCR.

Torque teno virus (TTV) is a group of chronically persisting viruses with a short circular DNA genome. TTV demonstrates a wide sequence diversity and a large majority of humans are chronically infected by one or more types of TTV. As TTV is ubiquitous, and viral replication correlates with immune status, TTV has been studied as a marker to assess global functional immune competence in transplant recipients. Most studies of the prevalence, amounts, and variation in TTV have been performed using PCR assays. We here present a comparison of the most frequently used quantitative PCR (qPCR) assay for TTV with shotgun metagenomic sequencing for detection and characterization of TTV in a cohort of pediatric cancer patients. The results show that TTV is more common than the qPCR assays indicate, and analysis of the TTV genome sequences indicate that a qPCR with primers and probe designed on a conserved region of the TTV genome may fail to detect some of the TTV strains found in this study.

No Correlation Between Nasopharyngeal Human Bocavirus 1 Genome Load and mRNA Detection or Serology in Adeno-/Tonsillectomy Patients.

Human bocavirus 1 (HBoV1) can persist in nasopharynx and tonsils. Using HBoV1 serology, reverse-transcription polymerase chain reaction (PCR) for detecting messenger RNA (mRNA) and quantitative PCR for HBoV1 genome load count, we studied to what extent the HBoV1 DNA loads in nasopharynx correlate with acute infection markers. Tonsillar tissue, nasopharyngeal aspirate, and serum were obtained from 188 elective adeno-/tonsillectomy patients. Relatively high loads of HBoV1 DNA were detected in the nasopharynx of 14 (7%) primarily asymptomatic subjects with negative mRNA and/or serodiagnostic results. Quantitative HBoV1 DNA PCR may have lower specificity than HBoV1 mRNA detection for diagnosing symptomatic infection.

The ketogenic diet influences taxonomic and functional composition of the gut microbiota in children with severe epilepsy.

The gut microbiota has been linked to various neurological disorders via the gut-brain axis. Diet influences the composition of the gut microbiota. The ketogenic diet (KD) is a high-fat, adequate-protein, low-carbohydrate diet established for treatment of therapy-resistant epilepsy in children. Its efficacy in reducing seizures has been confirmed, but the mechanisms remain elusive. The diet has also shown positive effects in a wide range of other diseases, including Alzheimer's, depression, autism, cancer, and type 2 diabetes. We collected fecal samples from 12 children with therapy-resistant epilepsy before starting KD and after 3 months on the diet. Parents did not start KD and served as diet controls. Applying shotgun metagenomic DNA sequencing, both taxonomic and functional profiles were established. Here we report that alpha diversity is not changed significantly during the diet, but differences in both taxonomic and functional composition are detected. Relative abundance of bifidobacteria as well as E. rectale and Dialister is significantly diminished during the intervention. An increase in relative abundance of E. coli is observed on KD. Functional analysis revealed changes in 29 SEED subsystems including the reduction of seven pathways involved in carbohydrate metabolism. Decomposition of these shifts indicates that bifidobacteria and Escherichia are important contributors to the observed functional shifts. As relative abundance of health-promoting, fiber-consuming bacteria becomes less abundant during KD, we raise concern about the effects of the diet on the gut microbiota and overall health. Further studies need to investigate whether these changes are necessary for the therapeutic effect of KD.

Tonsillar cytokine expression between patients with tonsillar hypertrophy and recurrent tonsillitis.

BACKGROUND: Tonsils provide an innovative in vivo model for investigating immune response to infections and allergens. However, data are scarce on the differences in tonsillar virus infections and immune responses between patients with tonsillar hypertrophy or recurrent tonsillitis. We investigated the differences in virus detection and T cell and interferon gene expression in patients undergoing tonsillectomy due to tonsillar hypertrophy or recurrent tonsillitis. METHODS: Tonsils of 89 surgical patients with tonsillar hypertrophy (n = 47) or recurrent tonsillitis (n = 42) were analysed. Patients were carefully characterized clinically. Standard questionnaire was used to asses preceding and allergy symptoms. Respiratory viruses were analysed in tonsils and nasopharynx by PCR. Quantitative real-time PCR was used to analyse intratonsillar gene expressions of IFN-α, IFN-ß, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-ß, FOXP3, GATA3, RORC2 and Tbet. RESULTS: Median age of the subjects was 15 years (range 2-60). Patients with tonsillar hypertrophy were younger, smoked less often, had less pollen allergy and had more adenovirus, bocavirus-1, coronavirus and rhinovirus in nasopharynx (all P < 0.05). Only bocavirus-1 was more often detected in hypertrophic tonsils (P < 0.05). In age-adjusted analysis, tonsillar hypertrophy was associated with higher mRNA expressions of IL-37 (P < 0.05). CONCLUSIONS: Intratonsillar T cell and interferon gene expressions appeared to be relatively stable for both tonsillar hypertrophy and recurrent tonsillitis. Of the studied cytokines, only newly discovered anti-inflammatory cytokine IL-37, was independently associated with tonsillar hypertrophy showing slightly stronger anti-inflammatory response in these patients.

Viremic co-infections in children with allogeneic haematopoietic stem cell transplantation are predominated by human polyomaviruses.

BACKGROUND: Viral infections remain the cause of key complications following haematopoietic stem cell transplantation (HSCT). The impact of multiple, concurrent viral reactivations/infections remains to be delineated. METHODS: The clinical correlates of single or multiple viremic infections following HSCT and especially the occurrence of respiratory viruses in the bloodstream were investigated. We retrospectively searched for 23 viruses in a total of 184 sera from 53 paediatric patients. The time-points of interest were pre-HSCT, one, two and three months post-HSCT, and at discharge or death. The viruses were analyzed by quantitative or qualitative PCR. RESULTS: Of the 53 patients, 13 (25%) had viraemias by multiple viruses and 27 (51%) by a single virus. Thirteen patients (25%) had no viruses detected by PCR during the study period. In the children with viremic co-infections, polyomaviruses predominated over herpes viruses. Nearly half the patients, 24/53 (45%) had a polyomavirus in their serum at one or more time-points. At 12/15 time-points and in 11/13 patients with co-infections polyomaviruses were involved, compared with 6/15 time-points and 6/13 patients for cytomegalovirus. Acute graft-versus-host disease (GvHD) and steroid use were significant risk factors for the viraemias caused by more than one virus. CONCLUSIONS: Viral co-detection is a common finding in children undergoing HSCT. With large-scale viral screening also viruses other than CMV could be found as potential pathogens. In this study, BKPyV predominated over CMV as a contributor in viraemias caused by multiple viruses in children receiving HSCT.

Polyomaviruses BK, JC, KI, WU, MC, and TS in children with allogeneic hematopoietic stem cell transplantation.

Timely and reliable detection of viruses is of key importance in early diagnosis of infection(s) following allogeneic HSCT. Among the immunocompetent, infections with BKPyV and JCPyV are mostly subclinical, while post-HSCT, the former may cause HC and the latter PML. The epidemiology and clinical impact of the newly identified KIPyV, WUPyV, MCPyV, and TSPyV in this context remain to be defined. To assess the incidence and clinical impact of BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, and TSPyV infections, we performed longitudinal molecular surveillance for DNAemias of these HPyVs among 53 pediatric HSCT recipients. Surveillance pre-HSCT and for three months post-HSCT revealed BKPyV DNAemia in 20 (38%) patients. Our data demonstrate frequent BKPyV DNAemia among pediatric patients with HSCT and the confinement of clinical symptoms to high copy numbers alone. MCPyV and JCPyV viremias occurred at low and TSPyV viremia at very low prevalences. KIPyV or WUPyV viremias were not demonstrable in this group of immunocompromised patients.

KI, WU, and Merkel cell polyomavirus DNA was not detected in guthrie cards of children who later developed acute lymphoblastic leukemia.

BACKGROUND: Neonatal dried blood spots (Guthrie cards) have been used to demonstrate a prenatal origin of clonal leukemia-specific genetic aberrations in several subgroups of childhood acute lymphoblastic leukemia (ALL). One hypothesis suggests that an infectious agent could initiate genetic transformation already in utero. In search for a possible viral agent, Guthrie cards were analyzed for the presence of 3 newly discovered polyomavirus Karolinska Institutet polymavirus (KIPyV), Washington University polyomavirus (WUPyV), and Merkel cell polyomavirus (MCPyV). METHODS: Guthrie cards from 50 children who later developed ALL and 100 matched controls were collected and analyzed by standard or real-time polymerase chain reaction for the presence of the VP1 region of KIPyV, WUPyV, and MCPyV, and the LT region for MCPyV. RESULTS AND CONCLUSIONS: DNA from KIPyV, WUPyV, and MCPyV was not detected in neonatal blood samples from children with ALL or controls. Prenatal infections with these viruses are not likely to be etiological drivers for childhood leukemogenesis.

Taxonomical developments in the family Polyomaviridae.

The Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV) has recommended several taxonomical revisions, as follows: The family Polyomaviridae, which is currently constituted as a single genus (Polyomavirus), will be comprised of three genera: two containing mammalian viruses and one containing avian viruses. The two mammalian genera will be designated Orthopolyomavirus and Wukipolyomavirus, and the avian genus will be named Avipolyomavirus. These genera will be created by the redistribution of species from the current single genus (Polyomavirus) and by the inclusion of several new species. In addition, the names of several species will be changed to reflect current usage.

An unbiased metagenomic search for infectious agents using monozygotic twins discordant for chronic fatigue.

BACKGROUND: Chronic fatigue syndrome is an idiopathic syndrome widely suspected of having an infectious or immune etiology. We applied an unbiased metagenomic approach to try to identify known or novel infectious agents in the serum of 45 cases with chronic fatigue syndrome or idiopathic chronic fatigue. Controls were the unaffected monozygotic co-twins of cases, and serum samples were obtained at the same place and time. RESULTS: No novel DNA or RNA viral signatures were confidently identified. Four affected twins and no unaffected twins evidenced viremia with GB virus C (8.9% vs. 0%, p = 0.019), and one affected twin had previously undetected hepatitis C viremia. An excess of GB virus C viremia in cases with chronic fatigue requires confirmation. CONCLUSIONS: Current, impairing chronic fatigue was not robustly associated with viremia detectable in serum.

Expression and serological characterization of polyomavirus WUPyV and KIPyV structural proteins.

The polyomaviruses WUPyV and KIPyV were recently discovered. We expressed their structural proteins VP1, VP2, and VP3, and the corresponding proteins of BKV and JCV, for immunoblotting of IgG antibodies from 115 wheezing young children and 25 asymptomatic adults. Furthermore, nasopharyngeal aspirates (NPA) and sera from the children were examined by PCR for viral DNA. The overlapping minor proteins VP2 and VP3 of WUPyV and KIPyV were more reactive in immunoblots than the major protein VP1; of 100 NPA PCR-negative wheezing children aged < or = 4 y, 31 (31%) and 31 (31%) were positive for WUPyV and KIPyV VP2/VP3, compared to only 3 (3%) and 5 (5%) for VP1, respectively. For comparison, the respective WUPyV and KIPyV IgG seroprevalences as determined by immunofluorescence assay (IFA) with nondenatured VP1 were 80% and 54%, respectively, among 50 NPA PCR-negative children aged < or = 2 y. This difference shows the importance of conformational VP1 antigenicity. Of the 25 adults, 52% and 68% were IgG-positive in immunoblots for VP2/VP3 of WUPyV and KIPyV, and 8% and 12% were for VP1, respectively. Of the 192 NPA samples studied by PCR, 7 (3.6%) were positive for WUPyV, and 3 (1.5%) were positive for KIPyV DNA. Unlike the NPA samples, none of the corresponding 443 sera contained WUPyV or KIPyV DNA. Together with the high VP2/VP3 IgG prevalence, this points to a paucity or brevity of KIPyV and WUPyV viremias among immunocompetent children. Our results indicate the significance of protein conformation in immunoreactivity of VP1, and show the antigenic importance of the WUPyV and KIPyV minor proteins VP2 and VP3. The high and rapidly increasing IgG prevalence rates observed in this study for WUPyV and KIPyV support the notion that these novel polyomaviruses are widespread and are acquired early in childhood.

Whole-genome characterization and genotyping of global WU polyomavirus strains.

Exploration of the genetic diversity of WU polyomavirus (WUV) has been limited in terms of the specimen numbers and particularly the sizes of the genomic fragments analyzed. Using whole-genome sequencing of 48 WUV strains collected in four continents over a 5-year period and 16 publicly available whole-genome sequences, we identified three main WUV clades and five subtypes, provisionally termed Ia, Ib, Ic, II, IIIa, and IIIb. Overall nucleotide variation was low (0 to 1.2%). The discriminatory power of the previous VP2 fragment typing method was found to be limited, and a new, larger genotyping region within the VP2/1 interface was proposed.

Respiratory viruses, a common microbiological finding in neutropenic children with fever.

BACKGROUND: Febrile neutropenia is a common complication in children undergoing chemotherapy for malignancies. A microbial agent is only identified in 15-30% of the fever episodes and corresponds mostly to bacterial findings. OBJECTIVE: To investigate viral infections as possible etiologic agents in episodes of febrile neutropenia. STUDY DESIGN: Nasopharyngeal aspirates (NPAs) from patients presenting with neutropenic fever at two pediatric oncology wards in Sweden and Australia were analyzed with a conventional virus-diagnostic approach and RT-PCR. Coupled blood samples were analyzed for the detection of CMV, EBV, adenovirus and erythrovirus. Bacterial blood culture was performed routinely. RESULTS: Conventional virus-diagnostic approach coupled to routinely performed bacterial analyzes revealed an infectious agent in 29% compared to 60% when using PCR. By adding PCR, a viral pathogen was detected in 46% of the NPAs and in 4% of the blood samples collected. In half of the patients with bacteremia, respiratory tract viruses were co-detected. CONCLUSION: Respiratory viruses were frequently detected in NPAs suggesting a significant role of viral infections in children presenting with neutropenic fever. The meaning of these findings needs to be further evaluated but has the potential to individualize infection treatment and to reduce the extensive use of antibiotics in immunocompromised children with neutropenia.

DNA from KI, WU and Merkel cell polyomaviruses is not detected in childhood central nervous system tumours or neuroblastomas.

BACKGROUND: BK and JC polyomaviruses (BKV and JCV) are potentially oncogenic and have in the past inconclusively been associated with tumours of the central nervous system (CNS), while BKV has been hinted, but not confirmed to be associated with neuroblastomas. Recently three new polyomaviruses (KIPyV, WUPyV and MCPyV) were identified in humans. So far KIPyV and WUPyV have not been associated to human diseases, while MCPyV was discovered in Merkel Cell carcinomas and may have neuroepithelial cell tropism. However, all three viruses can be potentially oncogenic and this compelled us to investigate for their presence in childhood CNS and neuroblastomas. METHODOLOGY: The presence of KI, WU and MCPyV DNA was analysed, by a joint WU and KI specific PCR (covering part of VP1) and by a MCPyV specific regular and real time quantitative PCR (covering part of Large T) in 25 CNS tumour biopsies and 31 neuroblastoma biopsies from the Karolinska University Hospital, Sweden. None of the three new human polyomaviruses were found to be associated with any of the tumours, despite the presence of PCR amplifiable DNA assayed by a S14 housekeeping gene PCR. CONCLUSION: In this pilot study, the presence of MCPyV, KI and WU was not observed in childhood CNS tumours and neuroblastomas. Nonetheless, we suggest that additional data are warranted in tumours of the central and peripheral nervous systems and we do not exclude that other still not yet detected polyomaviruses could be present in these tumours.

Merkel cell polyomavirus in respiratory tract secretions.

Merkel cell polyomavirus (MCPyV), associated with Merkel cell carcinoma, was detected in 27 of 635 nasopharyngeal aspirate samples by real-time PCR. MCPyV was more commonly found in adults than in children. Presence in the upper respiratory tract may be a general property of human PyVs.

A single-tube, real-time PCR assay for detection of the two newly characterized human KI and WU polyomaviruses.

BACKGROUND: Three new human polyomaviruses have been recently described, and investigating their in vivo biology and pathogenicity will require sensitive and rational detection assays. OBJECTIVES: To develop and evaluate a sensitive and rational assay for detection of the newly identified KI and WU polyomaviruses. STUDY DESIGN: A single-tube, dual-probe, real-time PCR assay for simultaneous detection and discrimination of KI and WU polyomaviruses was developed. RESULTS: The assay had near single-molecule sensitivity for both viruses and no cross-reactivity was observed. A panel of 637 nasopharyngeal aspirates was screened, resulting in a frequency of 1.4% for KIPyV and 1.3% for WUPyV. CONCLUSIONS: The dual-probe assay provides a rational approach for further studies of KIPyV and WUPyV.

Human bocavirus in children with acute lymphoblastic leukemia.

A new human parvovirus, human bocavirus, has recently been identified in respiratory secretions, feces and serum. It is associated with lower and most likely also upper respiratory tract infections. Most commonly reported symptoms are cough, rhinorrhea, expiratory wheezing and fever, and the virus is preferentially detected in young children. We report three children with acute lymphoblastic leukemia who had acute febrile episodes with concomitant detection of human bocavirus in their respiratory secretions. One of them had five consecutive febrile episodes during 6 months, all associated with the presence of human bocavirus at varying viral loads, suggesting prolonged shedding or reactivation of the virus.

Identification of a third human polyomavirus.

We have previously reported on a system for large-scale molecular virus screening of clinical samples. As part of an effort to systematically search for unrecognized human pathogens, the technology was applied for virus screening of human respiratory tract samples. This resulted in the identification of a previously unknown polyomavirus provisionally named KI polyomavirus. The virus is phylogenetically related to other primate polyomaviruses in the early region of the genome but has very little homology (<30% amino acid identity) to known polyomaviruses in the late region. The virus was found by PCR in 6 (1%) of 637 nasopharyngeal aspirates and in 1 (0.5%) of 192 fecal samples but was not detected in sets of urine and blood samples. Since polyomaviruses have oncogenic potential and may produce severe disease in immunosuppressed individuals, continued searching for the virus in different medical contexts is important. This finding further illustrates how unbiased screening of respiratory tract samples can be used for the discovery of diverse virus types.

Human monoclonal antibodies that react with the E2 glycoprotein of hepatitis C virus and possess neutralizing activity.

Active and/or passive immunoprophylaxis against hepatitis C virus (HCV) remain unachieved goals. Monoclonal antibodies might provide one approach to protection. We derived human monoclonal antibodies from the bone marrow of a patient with a well-controlled HCV infection of 22 years duration. Five distinct antibodies reactive with the E2 glycoprotein of the homologous 1a strain of HCV were recovered as antigen-binding fragments (FAbs). They demonstrated affinity constants as high as 2 nanomolar. "Neutralization of binding" titers paralleled the affinity constants. All five FAbs reacted with soluble E2 protein only in nonreducing gels, indicating that the relevant epitopes were conformational. The FAbs could be divided into two groups, based on competition analysis. Three of the FAbs neutralized the infectivity of pseudotyped virus particles (pp) bearing the envelope glycoproteins of the homologous HCV strain (genotype 1a). The three FAbs also neutralized genotype 1b pp and one also neutralized genotype 2a pp. In conclusion, one or more of these monoclonal antibodies may be useful in preventing infections by HCV belonging to genotype 1 or 2, the most medically important genotypes worldwide.

Recombinant human monoclonal antibodies against different conformational epitopes of the E2 envelope glycoprotein of hepatitis C virus that inhibit its interaction with CD81.

The antibody response to the envelope proteins of hepatitis C virus (HCV) may play an important role in controlling the infection. To allow molecular analyses of protective antibodies, we isolated human monoclonal antibodies to the E2 envelope glycoprotein of HCV from a combinatorial Fab library established from bone marrow of a chronically HCV-infected patient. Anti-E2 reactive clones were selected using recombinant E2 protein. The bone marrow donor carried HCV genotype 2b, and E2 used for selection was of genotype 1a. The antibody clones were expressed as Fab fragments in E. coli, and as Fab fragments and IgG1 in CHO cells. Seven different antibody clones were characterized, and shown to have high affinity for E2, genotype 1a. Three clones also had high affinity for E2 of genotype 1b. They all bind to conformation-dependent epitopes. Five clones compete for the same or overlapping binding sites, while two bind to one or two other epitopes of E2. Four clones corresponding to the different epitopes were tested as purified IgG1 for blocking the CD81-E2 interaction in vitro; all four were positive at 0.3-0.5 microg/ml. Thus, the present results suggest the existence of at least two conserved epitopes in E2 that mediate inhibition of the E2-CD81 interaction, of which one appeared immunodominant in this donor.