Human Protoparvoviruses

Next-generation sequencing and metagenomics have revolutionized the discovery of novel viruses. In recent years, three novel protoparvoviruses have been discovered in fecal samples of humans: bufavirus (BuV) in 2012, tusavirus (TuV) in 2014, and cutavirus (CuV) in 2016. BuV has since been studied the most, disclosing three genotypes that also represent serotypes. Besides one nasal sample, BuV DNA has been found exclusively in diarrheal feces, but not in non-diarrheal feces, suggesting a causal relationship. According to both geno- and seroprevalences, BuV appears to be the most common of the three novel protoparvoviruses, whereas TuV DNA has been found in only a single fecal sample, with antibody detection being equally rare. Moreover, the TuV sequence is closer to those of non-human protoparvoviruses, and so the evidence of TuV being a human virus is thus far insufficient. Interestingly, besides in feces, CuV has also been detected in skin biopsies of patients with cutaneous T-cell lymphoma and a patient with melanoma, while all other skin samples have tested PCR negative. Even if preliminary disease associations exist, the full etiological roles of these viruses in human disease are yet to be resolved.Peer reviewe

Microsphere-Based IgM and IgG Avidity Assays for Human Parvovirus B19, Human Cytomegalovirus, and Toxoplasma gondii

Human parvovirus B19 (here B19), human cytomegalovirus (HCMV), and Toxoplasma gondii infections during pregnancy can lead to severe complications. While traditional diagnosis of infections is mostly confined to one pathogen at a time, a multiplex array is a feasible alternative to improve diagnostic management and cost-efficiency. In the present study, for these three pathogens, we developed microsphere-based suspension immunoassays (SIAs) in multiplex and monoplex formats for the detection of antimicrobial IgM antibodies as well as corresponding chaotrope-based IgG avidity SIAs. We determined the diagnostic performances of the SIAs versus in-house and commercial reference assays using a panel of 318 serum samples from well-characterized clinical cohorts. All the newly developed assays exhibited excellent performance compared to the corresponding high-quality reference methods. The positive and negative percent agreements of the IgM SIAs in comparison with reference methods were 95 to 100% and 98 to 100%, and those of the IgG avidity SIAs were 92 to 100% and 95 to 100%, respectively. Kappa efficiency values between the SIAs and the corresponding reference assays were 0.91 to 1. Furthermore, with another panel comprising 391 clinical samples from individuals with primary infection by B19, HCMV, or T. gondii, the IgM SIAs were highly sensitive for the detection of acute infections, and the IgG avidity SIAs were highly specific for the separation of primary infections from past immunity. Altogether, the strategy of IgM multiplex screening followed by IgG avidity reflex testing can provide high-throughput and accurate means for the detection and stage determination of B19, HCMV, and T. gondii infections.IMPORTANCE Human parvovirus B19, human cytomegalovirus, and Toxoplasma gondii are ubiquitous pathogens. Their infections are often asymptomatic or mild in the general population yet may be transmitted from mother to fetus during pregnancy. Maternal infections by these pathogens can cause severe complications to the fetus or congenital abnormalities. As a rule, the risk of maternal transmission is critically related to the infection time; hence, it is important to determine when a pregnant woman has acquired the infection. In this study, we developed new diagnostic approaches for the timing of infections by three pathogens. All the new assays appeared to be highly sensitive and specific, providing powerful tools for medical diagnosis.Peer reviewe

Longitudinal assessment of the CXCL10 blood and urine concentration in kidney transplant recipients with BK polyomavirus replication—a retrospective study

In kidney transplant recipients (KTRs), BK polyomavirus (BKPyV) replication may progress to polyomavirus-associated nephropathy (PVAN). In this retrospective study, we assessed the chemokine CXCL10 in urine and blood samples consecutively acquired from 85 KTRs who displayed different stages of BKPyV replication and eventually developed PVAN. In parallel to progression toward PVAN, CXCL10 gradually increased in blood and urine, from baseline (prior to virus replication) to BKPyV DNAuria (median increase in blood: 42.15 pg/ml, P = 0.0156), from mere DNAuria to low- and high-level BKPyV DNAemia (median increase: 52.60 and 87.26 pg/ml, P = 0.0010 and P = 0.0002, respectively) and peaked with histologically confirmed PVAN (median increase: 145.00 pg/ml, P <0.0001). CXCL10 blood and urine levels significantly differed among KTRs with respect to simultaneous presence of human cytomegalovirus (P <0.001) as well as in relation to the clinical severity of respective BKPyV DNAemia episodes (P = 0.0195). CXCL-10 concentrations were particularly lower in KTRs in whom BKPyV DNAemia remained without clinical evidence for PVAN, as compared to individuals who displayed high decoy cell levels, decreased renal function and/or biopsy-proven PVAN (median blood concentration: 266.97 vs. 426.42 pg/ml, P = 0.0282). In conclusion, in KTRs CXCL10 rises in parallel to BKPyV replication and correlates with the gradual development of PVAN.Peer reviewe

Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials

Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials

Mononukleoosi

English summar

A hybrid pipeline for reconstruction and analysis of viral genomes at multi-organ level

Background: Advances in sequencing technologies have enabled the characterization of multiple microbial and host genomes, opening new frontiers of knowledge while kindling novel applications and research perspectives. Among these is the investigation of the viral communities residing in the human body and their impact on health and disease. To this end, the study of samples from multiple tissues is critical, yet, the complexity of such analysis calls for a dedicated pipeline. We provide an automatic and efficient pipeline for identification, assembly, and analysis of viral genomes that combines the DNA sequence data from multiple organs. TRACESPipe relies on cooperation among 3 modalities: compression-based prediction, sequence alignment, and de novo assembly. The pipeline is ultra-fast and provides, additionally, secure transmission and storage of sensitive data. Findings: TRACESPipe performed outstandingly when tested on synthetic and ex vivo datasets, identifying and reconstructing all the viral genomes, including those with high levels of single-nucleotide polymorphisms. It also detected minimal levels of genomic variation between different organs. Conclusions: TRACESPipe's unique ability to simultaneously process and analyze samples from different sources enables the evaluation of within-host variability. This opens up the possibility to investigate viral tissue tropism, evolution, fitness, and disease associations. Moreover, additional features such as DNA damage estimation and mitochondrial DNA reconstruction and analysis, as well as exogenous-source controls, expand the utility of this pipeline to other fields such as forensics and ancient DNA studies. TRACESPipe is released under GPLv3 and is available for free download at https://github.com/viromelab/tracespipe.Peer reviewe

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

Abstract 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

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

Copyright: This record is sourced from MEDLINE/PubMed, a database of the U.S. National Library of MedicineBACKGROUND: 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.Peer reviewe