Presence of herpesviruses, parvoviruses, and polyomaviruses in sinonasal lymphoma.

PURPOSE: Sinonasal lymphoma (SL) is a rare lymphatic neoplasm of the nasal cavities, paranasal sinuses and nasopharynx. Whereas some risk factors for SL subtypes have been identified, their aetiology is unknown. Along with other predisposing factors, the viral association of lymphomas, such as Epstein-Barr virus (EBV) and Burkitt and Hodgkin lymphomas, is well-established. Modern molecular biology techniques have enabled the discovery of novel human viruses, exemplified by the protoparvovirus cutavirus (CuV), associated with cutaneous T-cell lymphoma. These findings, and the anatomical location of the sinonasal tract with its rich microbiome and infectious agents, justify in-depth studies among SL. METHODS: We analysed the presence of 20 viruses of Orthoherpesviridae, Parvoviridae, and Polyomaviridae by qPCR in 24 SL tumours. We performed RNAscope in situ hybridisation (RISH) to localize the viruses. Parvovirus-specific IgG was analysed by enzyme immunoassay and targeted next-generation sequencing (NGS) was applied to detect CuV in plasma. RESULTS: We detected viral DNA in 15/24 (63%) tumours; nine of EBV, six of human herpesvirus (HHV) -7, four each of HHV-6B and parvovirus B19, two of cytomegalovirus, and one each of CuV and Merkel-cell polyomavirus. We found tumours with up to four viruses per tumour, and localized CuV and EBV DNAs by RISH. Two of the ten plasma samples exhibited CuV IgG, and one plasma sample demonstrated CuV viremia by NGS. CONCLUSION: Viruses were frequent findings in SL. The EBV detection rate was high in diffuse large B-cell lymphoma, and co-detections with other viruses were prevalent.

Herpesviruses, polyomaviruses, parvoviruses, papillomaviruses, and anelloviruses in vestibular schwannoma.

Etiology of vestibular schwannoma (VS) is unknown. Viruses can infect and reside in neural tissues for decades, and new viruses with unknown tumorigenic potential have been discovered. The presence of herpesvirus, polyomavirus, parvovirus, and anellovirus DNA was analyzed by quantitative PCR in 46 formalin-fixed paraffin-embedded VS samples. Five samples were analyzed by targeted next-generation sequencing. Viral DNA was detected altogether in 24/46 (52%) tumor samples, mostly representing anelloviruses (46%). Our findings show frequent persistence of anelloviruses, considered normal virome, in VS. None of the other viruses showed an extensive presence, thereby suggesting insignificant role in VS.

Unmasking the tissue-resident eukaryotic DNA virome in humans.

Little is known on the landscape of viruses that reside within our cells, nor on the interplay with the host imperative for their persistence. Yet, a lifetime of interactions conceivably have an imprint on our physiology and immune phenotype. In this work, we revealed the genetic make-up and unique composition of the known eukaryotic human DNA virome in nine organs (colon, liver, lung, heart, brain, kidney, skin, blood, hair) of 31 Finnish individuals. By integration of quantitative (qPCR) and qualitative (hybrid-capture sequencing) analysis, we identified the DNAs of 17 species, primarily herpes-, parvo-, papilloma- and anello-viruses (>80% prevalence), typically persisting in low copies (mean 540 copies/ million cells). We assembled in total 70 viral genomes (>90% breadth coverage), distinct in each of the individuals, and identified high sequence homology across the organs. Moreover, we detected variations in virome composition in two individuals with underlying malignant conditions. Our findings reveal unprecedented prevalences of viral DNAs in human organs and provide a fundamental ground for the investigation of disease correlates. Our results from post-mortem tissues call for investigation of the crosstalk between human DNA viruses, the host, and other microbes, as it predictably has a significant impact on our health.

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 (<250 copies/million cells) and longer PCR amplicons (>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.

Survey of Viral Reactivations in Elite Athletes: A Case-Control Study.

Exercise-induced immune perturbations have been proposed to increase susceptibility to viral infections. We investigated the replication of persisting viruses as indicators of immune function in elite cross-country skiers after ten months of sustained high-performance exercise. The viruses evaluated, nine human herpesviruses (HHVs) and torque teno virus (TTV), are typically restrained in health but replicate actively in immunosuppressed individuals. We collected sera from 27 Finnish elite cross-country skiers at the end of the competition's season and 27 matched controls who perform moderate exercise. We quantified all the HHVs and-TTV via highly sensitive qPCRs. To verify equal past exposures between the groups, we assessed the IgG antibody prevalences toward HHV-4 (Epstein-Barr virus, EBV) and HHV-5 (human cytomegalovirus, HCMV). We found equal TTV DNA prevalences in athletes (63%) and controls (63%) and loads with respective geometric means of 1.7 × 103 and 1.2 × 103 copies/mL of serum. Overall, the copy numbers were low and consistent with those of healthy individuals. Neither of the groups presented with herpesvirus viremia despite similar past exposures to HHVs (seroprevalences of EBV 70% vs. 78% and HCMV 52% vs. 44% in athletes and controls, respectively). We found no evidence of increased replication of persistent viruses in elite athletes, arguing against impaired viral immunity due to high-performance exercise.

Comparison of approaches for IgG avidity calculation and a new highly sensitive and specific method with broad dynamic range.

BACKGROUND: Antimicrobial IgG avidity is measured in the diagnosis of infectious disease, for dating of primary infection or immunization. It is generally determined by either of two approaches, termed here the avidity index (AI) or end-point ratio (EPR), which differ in complexity and workload. While several variants of these approaches have been introduced, little comparative information exists on their clinical utility. METHODS: This study was performed to systematically compare the performances of these approaches and to design a new sensitive and specific calculation method, for easy implementation in the laboratory. The avidities obtained by AI, EPR, and the newly developed approach were compared, across parvovirus B19, cytomegalovirus, Toxoplasma gondii, rubella virus, and Epstein-Barr virus panels comprising 460 sera from individuals with a recent primary infection or long-term immunity. RESULTS: With optimal IgG concentrations, all approaches performed equally, appropriately discriminating primary infections from past immunity (area under the receiver operating characteristic curve (AUC) 0.93-0.94). However, at lower IgG concentrations, the avidity status (low, borderline, high) changed in 17% of samples using AI (AUC 0.88), as opposed to 4% using EPR (AUC 0.91) and 6% using the new method (AUC 0.93). CONCLUSIONS: The new method measures IgG avidity accurately, in a broad range of IgG levels, while the popular AI approach calls for a sufficiently high antibody concentration.

The Human Bone Marrow Is Host to the DNAs of Several Viruses.

The long-term impact of viruses residing in the human bone marrow (BM) remains unexplored. However, chronic inflammatory processes driven by single or multiple viruses could significantly alter hematopoiesis and immune function. We performed a systematic analysis of the DNAs of 38 viruses in the BM. We detected, by quantitative PCRs and next-generation sequencing, viral DNA in 88.9% of the samples, up to five viruses in one individual. Included were, among others, several herpesviruses, hepatitis B virus, Merkel cell polyomavirus and, unprecedentedly, human papillomavirus 31. Given the reactivation and/or oncogenic potential of these viruses, their repercussion on hematopoietic and malignant disorders calls for careful examination. Furthermore, the implications of persistent infections on the engraftment, regenerative capacity, and outcomes of bone marrow transplantation deserve in-depth evaluation.

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.

The landscape of persistent human DNA viruses in femoral bone.

The imprints left by persistent DNA viruses in the tissues can testify to the changes driving virus evolution as well as provide clues on the provenance of modern and ancient humans. However, the history hidden in skeletal remains is practically unknown, as only parvovirus B19 and hepatitis B virus DNA have been detected in hard tissues so far. Here, we investigated the DNA prevalences of 38 viruses in femoral bone of recently deceased individuals. To this end, we used quantitative PCRs and a custom viral targeted enrichment followed by next-generation sequencing. The data was analyzed with a tailor-made bioinformatics pipeline. Our findings revealed bone to be a much richer source of persistent DNA viruses than earlier perceived, discovering ten additional ones, including several members of the herpes- and polyomavirus families, as well as human papillomavirus 31 and torque teno virus. Remarkably, many of the viruses found have oncogenic potential and/or may reactivate in the elderly and immunosuppressed individuals. Thus, their persistence warrants careful evaluation of their clinical significance and impact on bone biology. Our findings open new frontiers for the study of virus evolution from ancient relics as well as provide new tools for the investigation of human skeletal remains in forensic and archaeological contexts.

HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses.

Infections with the nine human herpesviruses (HHVs) are globally prevalent and characterized by lifelong persistence. Reactivations can potentially manifest as life-threatening conditions for which the demonstration of viral DNA is essential. In the present study, we developed HERQ-9, a pan-HHV quantitative PCR designed in triplex reactions to differentiate and quantify each of the HHV-DNAs: (i) herpes simplex viruses 1 and 2 and varicella-zoster virus; (ii) Epstein-Barr virus, human cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus; and (iii) HHV-6A, -6B, and -7. The method was validated with prequantified reference standards as well as with mucocutaneous swabs and cerebrospinal fluid, plasma, and tonsillar tissue samples. Our findings highlight the value of multiplexing in the diagnosis of many unsuspected, yet clinically relevant, herpesviruses. In addition, we report here frequent HHV-DNA co-occurrences in clinical samples, including some previously unknown. HERQ-9 exhibited high specificity and sensitivity (LOD95s of ∼10 to ∼17 copies/reaction), with a dynamic range of 101 to 106 copies/µl. Moreover, it performed accurately in the coamplification of both high- and low-abundance targets in the same reaction. In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance to clinical management, the method is valuable for the assessment of hitherto-unexplored synergistic effects of herpesvirus coinfections. Furthermore, its high sensitivity enables studies on the human virome, often dealing with minute quantities of persisting HHVs.IMPORTANCE By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.

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.

Extinct type of human parvovirus B19 persists in tonsillar B cells.

Parvovirus B19 (B19V) DNA persists lifelong in human tissues, but the cell type harbouring it remains unclear. We here explore B19V DNA distribution in B, T and monocyte cell lineages of recently excised tonsillar tissues from 77 individuals with an age range of 2-69 years. We show that B19V DNA is most frequent and abundant among B cells, and within them we find a B19V genotype that vanished from circulation >40 years ago. Since re-infection or re-activation are unlikely with this virus type, this finding supports the maintenance of pathogen-specific humoral immune responses as a consequence of B-cell long-term survival rather than continuous replenishment of the memory pool. Moreover, we demonstrate the mechanism of B19V internalization to be antibody dependent in two B-cell lines as well as in ex vivo isolated tonsillar B cells. This study provides direct evidence for a cell type accountable for B19V DNA tissue persistence.

17th Century Variola Virus Reveals the Recent History of Smallpox.

Smallpox holds a unique position in the history of medicine. It was the first disease for which a vaccine was developed and remains the only human disease eradicated by vaccination. Although there have been claims of smallpox in Egypt, India, and China dating back millennia [1-4], the timescale of emergence of the causative agent, variola virus (VARV), and how it evolved in the context of increasingly widespread immunization, have proven controversial [4-9]. In particular, some molecular-clock-based studies have suggested that key events in VARV evolution only occurred during the last two centuries [4-6] and hence in apparent conflict with anecdotal historical reports, although it is difficult to distinguish smallpox from other pustular rashes by description alone. To address these issues, we captured, sequenced, and reconstructed a draft genome of an ancient strain of VARV, sampled from a Lithuanian child mummy dating between 1643 and 1665 and close to the time of several documented European epidemics [1, 2, 10]. When compared to vaccinia virus, this archival strain contained the same pattern of gene degradation as 20th century VARVs, indicating that such loss of gene function had occurred before ca. 1650. Strikingly, the mummy sequence fell basal to all currently sequenced strains of VARV on phylogenetic trees. Molecular-clock analyses revealed a strong clock-like structure and that the timescale of smallpox evolution is more recent than often supposed, with the diversification of major viral lineages only occurring within the 18th and 19th centuries, concomitant with the development of modern vaccination.

Microsphere-based antibody assays for human parvovirus B19V, CMV and T. gondii.

BACKGROUND: Human parvovirus B19 (B19V), cytomegalovirus (CMV) and Toxoplasma gondii (T. gondii) may cause intrauterine infections with potentially severe consequences to the fetus. Current serodiagnosis of these infections is based on detection of antibodies most often by EIA and individually for each pathogen. We developed singleplex and multiplex microsphere-based Suspension Immuno Assays (SIAs) for the simultaneous detection of IgG antibodies against B19V, CMV and T. gondii. METHODS: We tested the performances of SIAs as compared to in-house and commercial reference assays using serum samples from well-characterized cohorts. RESULTS: The IgG SIAs for CMV and T. gondii showed good concordance with the corresponding Vidas serodiagnostics. The B19V IgG SIA detected IgG in all samples collected >10 days after onset of symptoms and showed high concordance with EIAs (in-house and Biotrin). The serodiagnostics for these three pathogens performed well in multiplex format. CONCLUSIONS: We developed singleplex and multiplex IgG SIAs for the detection of anti-B19V, -CMV and -T. gondii antibodies. The SIAs were highly sensitive and specific, and had a wide dynamic range. These components thus should be suitable for construction of a multiplex test for antibody screening during pregnancy.

Granzyme B mediated function of Parvovirus B19-specific CD4(+) T cells.

A novel conception of CD4(+) T cells with cytolytic potential (CD4(+) CTL) is emerging. These cells appear to have a part in controlling malignancies and chronic infections. Human parvovirus B19 can cause a persistent infection, yet no data exist on the presence of B19-specific CD4(+) CTLs. Such cells could have a role in the pathogenesis of some autoimmune disorders reported to be associated with B19. We explored the cytolytic potential of human parvovirus B19-specific T cells by stimulating peripheral blood mononuclear cell (PBMC) with recombinant B19-VP2 virus-like particles. The cytolytic potential was determined by enzyme immunoassay-based quantitation of granzyme B (GrB) and perforin from the tissue culture supernatants, by intracellular cytokine staining (ICS) and by detecting direct cytotoxicity. GrB and perforin responses with the B19 antigen were readily detectable in B19-seropositive individuals. T-cell depletion, HLA blocking and ICS experiments showed GrB and perforin to be secreted by CD4(+) T cells. CD4(+) T cells with strong GrB responses were found to exhibit direct cytotoxicity. As anticipated, ICS of B19-specific CD4(+) T cells showed expected co-expression of GrB, perforin and interferon gamma (IFN-γ). Unexpectedly, also a strong co-expression of GrB and interleukin 17 (IL-17) was detected. These cells expressed natural killer (NK) cell surface marker CD56, together with the CD4 surface marker. To our knowledge, this is the first report on virus-specific CD4(+) CTLs co-expressing CD56 antigen. Our results suggest a role for CD4(+) CTL in B19 immunity. Such cells could function within both immune regulation and triggering of autoimmune phenomena such as systemic lupus erythematosus (SLE) or rheumatoid arthritis.

Human serum protein enhances HIV-1 replication and up-regulates the transcription factor AP-1.

In vitro studies on HIV (HIV-1) replication and neutralization are usually performed in human cell cultures supplemented with FBS instead of human serum (HS). Here we show that in contrast to FBS, addition of increasing amounts of human serum from noninfected donors to the cell culture directly correlates with an increase in HIV-1 replication in vitro. This effect is independent of cell line, virus strain, or batch of pooled human serum used. We found that human serum affects viral transcription in a dose-dependent manner by activating the activator protein-1 (AP-1) member proteins c-FOS, JunD, and JunB in TZM-bl cells. Analysis of the human serum component responsible for this effect indicates that it is a protein having a molecular mass between 250 and 300 kDa. This serum protein, HIV-1 enhancing serum protein (HESP), might promote viral transcription in vivo and consequently play a role in disease progression.

HIV inhibition by CD4 and CCR5-derived glycopeptides.

We have previously proposed a method by which natural antibodies can be redirected toward a known pathogen. We could show that CD4-derived peptides coupled to the galα1,3gal sugar moiety, a specificity held by natural antibodies, were able to neutralize HIV. Importantly, the antibody-peptide-antigen complexes activated the innate immune system through the Fc parts of the natural antibody. We now tested these peptides for their effectiveness on primary isolates and included sequence variations to increase their binding affinity. In addition, we evaluated three new CCR5-derived peptides. All peptides were tested for neutralization of six primary HIV-1 isolates. When testing three of the previously published glycopeptides we found that 10 to 100 times higher concentrations were needed to achieve the same neutralization of primary isolates. We found that the modifications of the CD4 glycopeptides modestly improved the neutralization of HIV-1. The modified CD4 and the CCR5 glycopeptides neutralized HIV-1 strains from different patients and of different subtypes. Notably, the combination of CD4 and CCR5 glycopeptides enhanced the neutralization potential as compared to the single peptides. A combination of CD4- and CCR5-galα1,3gal-linked peptides redirected natural antibodies to neutralize primary isolates of HIV-1, although less efficiently than laboratory-adapted strains. This might represent a new and valuable tool to block the entry of HIV into susceptible cells.

Neutralization of HIV-1 by redirection of natural antibodies.

The great variability and high glycosylation of gp120 poses a great challenge for the design of a functional immune therapy. The binding region of the CD4 receptor to gp120, however, is well conserved and may constitute a target to limit viral entry and infectivity. Our strategy consists in using a preexisting pool of natural antibodies directed toward the gal(alpha1,3)gal disaccharide and to redirect it to HIV. We here show that using CD4-derived, gp120-binding, synthetic peptides chemically linked to gal(alpha1,3)gal can redirect these natural antibodies and improve the HIV-1 neutralizing activity of the CD4-derived peptides in vitro. Importantly, the binding of the CD4-gal(alpha1,3)gal peptides to HIV-1-infected cells conferred antibody-dependent cellular cytotoxicity after the addition of human sera. Thus, the temporary redirection of naturally occurring antibodies and their biological activities to a new antigen represents a completely new way of targeting a human disease.