Widespread Horizontal Gene Transfer Among Animal Viruses.

The initial objective of this study was to shed light on the evolution of small DNA tumor viruses by analyzing de novo assemblies of publicly available deep sequencing datasets. The survey generated a searchable database of contig snapshots representing more than 100,000 Sequence Read Archive records. Using modern structure-aware search tools, we iteratively broadened the search to include an increasingly wide range of other virus families. The analysis revealed a surprisingly diverse range of chimeras involving different virus groups. In some instances, genes resembling known DNA-replication modules or known virion protein operons were paired with unrecognizable sequences that structural predictions suggest may represent previously unknown replicases and novel virion architectures. Discrete clades of an emerging group called adintoviruses were discovered in datasets representing humans and other primates. As a proof of concept, we show that the contig database is also useful for discovering RNA viruses and candidate archaeal phages. The ancillary searches revealed additional examples of chimerization between different virus groups. The observations support a gene-centric taxonomic framework that should be useful for future virus-hunting efforts.

A phase III randomized crossover trial of plerixafor versus G-CSF for treatment of WHIM syndrome.

BACKGROUNDWarts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is a kind of neutropenia caused by neutrophil retention in bone marrow and in WHIM syndrome is associated with lymphopenia and monocytopenia. The CXCR4 antagonist plerixafor mobilizes leukocytes to the blood; however, its safety and efficacy in WHIM syndrome are undefined.METHODSIn this investigator-initiated, single-center, quadruple-masked phase III crossover trial, we compared the total infection severity score (TISS) as the primary endpoint in an intent-to-treat manner in 19 patients with WHIM who each received 12 months treatment with plerixafor and 12 months treatment with granulocyte CSF (G-CSF, the standard of care for severe congenital neutropenia). The treatment order was randomized for each patient.RESULTSPlerixafor was nonsuperior to G-CSF for TISS (P = 0.54). In exploratory endpoints, plerixafor was noninferior to G-CSF for maintaining neutrophil counts of more than 500 cells/µL (P = 0.023) and was superior to G-CSF for maintaining lymphocyte counts above 1,000 cells/µL (P < 0.0001). Complete regression of a subset of large wart areas occurred on plerixafor in 5 of 7 patients with major wart burdens at baseline. Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life or the incidence of drug failure or serious adverse events.CONCLUSIONPlerixafor was not superior to G-CSF in patients with WHIM for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results support continued study of plerixafor as a potential treatment for WHIM syndrome.TRIAL REGISTRATIONClinicaltrials.gov NCT02231879.FUNDINGThis study was funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

A multivalent polyomavirus vaccine elicits durable neutralizing antibody responses in macaques.

In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals. Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomavirus vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of ≥ 25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN.

Adintoviruses: a proposed animal-tropic family of midsize eukaryotic linear dsDNA (MELD) viruses.

Polintons (also known as Mavericks) were initially identified as a widespread class of eukaryotic transposons named for their hallmark type B DNA polymerase and retrovirus-like integrase genes. It has since been recognized that many polintons encode possible capsid proteins and viral genome-packaging ATPases similar to those of a diverse range of double-stranded DNA viruses. This supports the inference that at least some polintons are actually viruses capable of cell-to-cell spread. At present, there are no polinton-associated capsid protein genes annotated in public sequence databases. To rectify this deficiency, we used a data-mining approach to investigate the distribution and gene content of polinton-like elements and related DNA viruses in animal genomic and metagenomic sequence datasets. The results define a discrete family-like clade of viruses with two genus-level divisions. We propose the family name Adintoviridae, connoting similarities to adenovirus virion proteins and the presence of a retrovirus-like integrase gene. Although adintovirus-class PolB sequences were detected in datasets for fungi and various unicellular eukaryotes, sequences resembling adintovirus virion proteins and accessory genes appear to be restricted to animals. Degraded adintovirus sequences are endogenized into the germlines of a wide range of animals, including humans.

Histone Modifications in Papillomavirus Virion Minichromosomes.

An unusual feature of papillomaviruses is that their genomes are packaged into virions along with host histones. Viral minichromosomes were visualized as "beads on a string" by electron microscopy in the 1970s but, to date, little is known about the posttranslational modifications of these histones. To investigate this, we analyzed the histone modifications in HPV16/18 quasivirions, wart-derived bovine papillomavirus (BPV1), and wart-derived human papillomavirus type 1 (HPV1) using quantitative mass spectrometry. The chromatin from all three virion samples had abundant posttranslational modifications (acetylation, methylation, and phosphorylation). These histone modifications were verified by acid urea polyacrylamide electrophoresis and immunoblot analysis. Compared to matched host cell controls, the virion minichromosome was enriched in histone modifications associated with active chromatin and depleted for those commonly found in repressed chromatin. We propose that the viral minichromosome acquires specific histone modifications late in infection that are coupled to the mechanisms of viral replication, late gene expression, and encapsidation. We predict that, in turn, these same modifications benefit early stages of infection by helping to evade detection, promoting localization of the viral chromosome to beneficial regions of the nucleus, and promoting early transcription and replication.IMPORTANCE A relatively unique feature of papillomaviruses is that the viral genome is associated with host histones inside the virion. However, little is known about the nature of the epigenome within papillomavirions or its biological relevance to the infectious viral cycle. Here, we define the epigenetic signature of the H3 and H4 histones from HPV16 virions generated in cell culture and native human papillomavirus type 1 (HPV1) and bovine papillomavirus 1 (BPV1) virions isolated from bovine and human wart tissue. We show that native virions are enriched in posttranslational modifications associated with active chromatin and depleted with those associated with repressed chromatin compared to cellular chromatin. Native virions were also enriched in the histone variant H3.3 compared to the canonical histone H3.1. We propose that the composition of virion-packaged chromatin reflects the late stages of the viral life cycle and promotes the early stages of infection by being primed for viral transcription.

Trichodysplasia spinulosa in a child: Identification of trichodysplasia spinulosa-associated polyomavirus in skin, serum, and urine.

A 6-year-old girl with a history of chronic immunosuppression following small bowel and colon transplantation for tufting enteropathy presented with a diffuse, facial-predominant eruption composed of pink-to-skin-colored papules with central white dystrophic spicules. Histology from a punch biopsy and polymerase chain reaction (PCR) from plucked spicules confirmed a diagnosis of trichodysplasia spinulosa (TS). Additional molecular studies identified several strains of the trichodysplasia spinulosa-associated polyomavirus infecting multiple tissues of the patient, confirming the systemic nature of trichodysplasia spinulosa infections.

Plerixafor for the Treatment of WHIM Syndrome.

WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis), a primary immunodeficiency disorder involving panleukopenia, is caused by autosomal dominant gain-of-function mutations in CXC chemokine receptor 4 (CXCR4). Myelokathexis is neutropenia caused by neutrophil retention in bone marrow. Patients with WHIM syndrome are often treated with granulocyte colony-stimulating factor (G-CSF), which can increase neutrophil counts but does not affect cytopenias other than neutropenia. In this investigator-initiated, open-label study, three severely affected patients with WHIM syndrome who could not receive G-CSF were treated with low-dose plerixafor, a CXCR4 antagonist, for 19 to 52 months. Myelofibrosis, panleukopenia, anemia, and thrombocytopenia were ameliorated, the wart burden and frequency of infection declined, human papillomavirus-associated oropharyngeal squamous-cell carcinoma stabilized, and quality of life improved markedly. Adverse events were mainly infections attributable to the underlying immunodeficiency. One patient died from complications of elective reconstructive surgery. (Funded by the National Institutes of Health.).

Development and evaluation of a BK polyomavirus serotyping assay using Luminex technology.

BACKGROUND: The BK polyomavirus (BKPyV) is subdivided into four genotypes. The consequences of each genotype and of donor-recipient genotype (mis)match for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant recipients (KTRs) are unknown. OBJECTIVES: To develop and evaluate a genotype-specific IgG antibody-based BKPyV serotyping assay, in order to classify kidney transplant donors and recipients accordingly. STUDY DESIGN: VP1 antigens of six BKPyV variants (Ib1, Ib2, Ic, II, III and IV) were expressed as recombinant glutathione-s-transferase-fusion proteins and coupled to fluorescent Luminex beads. Sera from 87 healthy blood donors and 39 KTRs were used to analyze seroreactivity and serospecificity against the different BKPyV genotypes. Six sera with marked BKPyV serotype profiles were analyzed further for genotype-specific BKPyV pseudovirus neutralizing capacity. RESULTS: Seroreactivity was observed against all genotypes, with seropositivity rates above 77% comparable for KTRs and blood donors. Strong cross-reactivity (r > 0.8) was observed among genotype I subtypes, and among genotypes II, III and IV. Seroresponses against genotypes I and IV seemed genuine, while those against II and III could be out(cross)competed. GMT (Luminex) and IC50 (neutralization assay) values showed good agreement in determining the genotype with the strongest seroresponse within an individual. CONCLUSIONS: Despite some degree of cross-reactivity, this serotyping assay seems a useful tool to identify the main infecting BKPyV genotype within a given individual. This information, which cannot be obtained otherwise from nonviremic/nonviruric individuals, could provide valuable information regarding the prevalent BKPyV genotype in kidney donors and recipients and warrants further study.

Metagenomic Discovery of 83 New Human Papillomavirus Types in Patients with Immunodeficiency.

Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus Gammapapillomavirus were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus Betapapillomavirus. Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts.IMPORTANCE Although some members of the viral family Papillomaviridae cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed Gammapapillomavirus (Gamma) virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known Gamma HPV types and suggest that WHIM syndrome patients are uniquely susceptible to Gamma HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual Gamma HPV skin warts observed in WHIM patients.

Characterization of BK Polyomaviruses from Kidney Transplant Recipients Suggests a Role for APOBEC3 in Driving In-Host Virus Evolution.

BK polyomavirus (BKV) frequently causes nephropathy (BKVN) in kidney transplant recipients (KTRs). BKV has also been implicated in the etiology of bladder and kidney cancers. We characterized BKV variants from two KTRs who developed BKVN followed by renal carcinoma. Both patients showed a swarm of BKV sequence variants encoding non-silent mutations in surface loops of the viral major capsid protein. The temporal appearance and disappearance of these mutations highlights the intra-patient evolution of BKV. Some of the observed mutations conferred resistance to antibody-mediated neutralization. The mutations also modified the spectrum of receptor glycans engaged by BKV during host cell entry. Intriguingly, all observed mutations were consistent with DNA damage caused by antiviral APOBEC3 cytosine deaminases. Moreover, APOBEC3 expression was evident upon immunohistochemical analysis of renal biopsies from KTRs. These results provide a snapshot of in-host BKV evolution and suggest that APOBEC3 may drive BKV mutagenesis in vivo.

Infectious Entry and Neutralization of Pathogenic JC Polyomaviruses.

Progressive multifocal leukoencephalopathy (PML) is a lethal brain disease caused by uncontrolled replication of JC polyomavirus (JCV). JCV strains recovered from the brains of PML patients carry mutations that prevent the engagement of sialylated glycans, which are thought to serve as receptors for the infectious entry of wild-type JCV. In this report, we show that non-sialylated glycosaminoglycans (GAGs) can serve as alternative attachment receptors for the infectious entry of both wild-type and PML mutant JCV strains. After GAG-mediated attachment, PML mutant strains engage non-sialylated non-GAG co-receptor glycans, such as asialo-GM1. JCV-neutralizing monoclonal antibodies isolated from patients who recovered from PML appear to block infection by preventing the docking of post-attachment co-receptor glycans in an apical pocket of the JCV major capsid protein. Identification of the GAG-dependent/sialylated glycan-independent alternative entry pathway should facilitate the development of infection inhibitors, including recombinant neutralizing antibodies.

Human polyomavirus 6 and 7 are associated with pruritic and dyskeratotic dermatoses.

BACKGROUND: Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, has been linked to a pruritic skin eruption of immunosuppression. OBJECTIVE: We determined whether biopsy specimens showing a characteristic pattern of dyskeratosis and parakeratosis might be associated with polyomavirus infection. METHODS: We screened biopsy specimens showing "peacock plumage" histology by polymerase chain reaction for HPyVs. Cases positive for HPyV6 or HPyV7 were then analyzed by immunohistochemistry, electron microscopy, immunofluorescence, quantitative polymerase chain reaction, and complete sequencing, including unbiased, next-generation sequencing. RESULTS: We identified 3 additional cases of HPyV6 or HPyV7 skin infections. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 primarily infects keratinocytes. High viral loads in lesional skin compared with normal-appearing skin and the identification of intact virions by both electron microscopy and next-generation sequencing support a role for active viral infections in these skin diseases. LIMITATION: This was a small case series of archived materials. CONCLUSION: We have found that HPyV6 and HPyV7 are associated with rare, pruritic skin eruptions with a distinctive histologic pattern and describe this entity as "HPyV6- and HPyV7-associated pruritic and dyskeratotic dermatoses."

The Ancient Evolutionary History of Polyomaviruses.

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

JC polyomavirus mutants escape antibody-mediated neutralization.

JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

Hamburger polyomaviruses.

Epidemiological studies have suggested that consumption of beef may correlate with an increased risk of colorectal cancer. One hypothesis to explain this proposed link might be the presence of a carcinogenic infectious agent capable of withstanding cooking. Polyomaviruses are a ubiquitous family of thermostable non-enveloped DNA viruses that are known to be carcinogenic. Using virion enrichment, rolling circle amplification (RCA) and next-generation sequencing, we searched for polyomaviruses in meat samples purchased from several supermarkets. Ground beef samples were found to contain three polyomavirus species. One species, bovine polyomavirus 1 (BoPyV1), was originally discovered as a contaminant in laboratory FCS. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus, both of which are carcinogenic in their native hosts. A third species, BoPyV3, is related to human polyomaviruses 6 and 7. Examples of additional DNA virus families, including herpesviruses, adenoviruses, circoviruses and gyroviruses were also detected either in ground beef samples or in comparison samples of ground pork and ground chicken. The results suggest that the virion enrichment/RCA approach is suitable for random detection of essentially any DNA virus with a detergent-stable capsid. It will be important for future studies to address the possibility that animal viruses commonly found in food might be associated with disease.

Genome Sequence of a Fish-Associated Polyomavirus, Black Sea Bass (Centropristis striata) Polyomavirus 1.

All known polyomaviruses are associated with mammals or birds. Using virion enrichment, random-primed rolling circle amplification, and deep sequencing, we identified a polyomavirus associated with black sea bass (Centropristis striata). The virus has a variety of novel genetic features, suggesting a long evolutionary separation from polyomaviruses of terrestrial animals.

Human polyomavirus 7-associated pruritic rash and viremia in transplant recipients.

Human polyomavirus 7 (HPyV7) is one of 11 HPyVs recently discovered through genomic sequencing technologies. Two lung transplant recipients receiving immunosuppressive therapy developed pruritic, brown plaques on the trunk and extremities showing a distinctive epidermal hyperplasia with virus-laden keratinocytes containing densely packed 36-45-nm icosahedral capsids. Rolling circle amplification and gradient centrifugation testing were positive for encapsidated HPyV7 DNA in skin and peripheral blood specimens from both patients, and HPyV7 early and capsid proteins were abundantly expressed in affected tissues. We describe for the first time that HPyV7 is associated with novel pathogenicity in some immunosuppressed individuals.

Detection of human polyomavirus 7 in human thymic epithelial tumors.

INTRODUCTION: Although the molecular genetics possibly underlying the pathogenesis of human thymoma have been extensively studied, its etiology remains poorly understood. Because murine polyomavirus consistently induces thymomas in mice, we assessed the presence of the novel human polyomavirus 7 (HPyV7) in human thymic epithelial tumors. METHODS: HPyV7-DNA Fluorescence in situ hybridization (FISH), DNA-polymerase chain reaction (PCR), and immunohistochemistry (IHC) were performed in 37 thymomas. Of these, 26 were previously diagnosed with myasthenia gravis (MG). In addition, 20 thymic hyperplasias and 20 fetal thymic tissues were tested. RESULTS: HPyV7-FISH revealed specific nuclear hybridization signals within the neoplastic epithelial cells of 23 thymomas (62.2%). With some exceptions, the HPyV7-FISH data correlated with the HPyV7-DNA PCR. By IHC, large T antigen expression of HPyV7 was detected, and double staining confirmed its expression in the neoplastic epithelial cells. Eighteen of the 26 MG-positive and 7 of the 11 MG-negative thymomas were HPyV7-positive. Of the 20 hyperplastic thymi, 40% were HPyV7-positive by PCR as confirmed by FISH and IHC in the follicular lymphocytes. All 20 fetal thymi tested HPyV7-negative. CONCLUSIONS: The presence of HPyV7-DNA and large T antigen expression in the majority of thymomas possibly link HPyV7 to human thymomagenesis. Further investigations are needed to elucidate the possible associations of HPyV7 and MG.