Joachim E. Kühn

A Novel Human Polyomavirus Closely Related to the African Green Monkey-Derived Lymphotropic Polyomavirus

ABSTRACT We identified a novel human polyomavirus from a kidney transplant patient under immunosuppressive treatment, by use of a generic PCR. The genome of the virus was completely amplified and sequenced. In phylogenetic analyses, it appeared as the closest relative to the African green monkey-derived lymphotropic polyomavirus (LPV). Further investigation of clinical samples from immunocompromised patients with specific nested PCR revealed additional positive samples, indicating that the virus naturally infects humans. The virus was tentatively named human polyomavirus 9 (HPyV9). The previously observed seroreactivity to LPV in human populations might find a partial explanation in the circulation of HPyV9.

Therapeutic Inflammatory Monocyte Modulation Using Immune-Modifying Microparticles

Negatively charged immune-modifying microparticles bind to the scavenger receptor MARCO on inflammatory monocytes, resulting in their apoptosis and reduced inflammatory damage in a range of diseases. A New Frontier in Immune Modulation Inflammatory monocytes markedly potentiate the immune pathology observed in many diseases, yet no therapy exists that specifically inhibits these cells. The therapeutic accessibility of monocytes in the bloodstream and their inherent propensity to engulf particulate material suggest that highly negatively charged microparticles might provide a readily translatable solution to this problem. These microparticles, referred to as immune-modifying microparticles (IMPs), may be derived from numerous compounds, including the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA-IMP), already used in humans for inter alia dissolvable sutures. Getts et al. now show that upon infusion, IMPs bind to a receptor with a positive domain on inflammatory monocytes, resulting in monocyte sequestration in the spleen and apoptosis through a similar pathway observed for senescing leukocytes. This safe monocyte clearance pathway culminated in substantially reduced inflammatory tissue damage in mouse models of West Nile virus encephalitis, experimental autoimmune encephalomyelitis, peritonitis, colitis, and myocardial infarction. Together, the data suggest that IMPs could transform the treatment of acute inflammation. Indeed, phase 1/2 testing is planned to begin in 2014, with rapid translation supported by the availability of clinical-grade PLGA. Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3–mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate–induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis.

A competitive nested PCR-temperature gradient gel electrophoresis protocol (nPCR/TGGE) has been established for the quantification of human cytomegalovirus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard (st) and separation of st and wild-type (wt) amplimers by temperature gradient gel electrophoresis (TGGE). The number of HCMV target sequences could be precisely determined within wt/st ratios of 0.1 to 10. With 50 copies of the st sequence the detection limit of nPCR/TGGE was found to be five to 10 copies of the target sequence. Effects of sample preparation on quantitative HCMV PCR were minimized by the additional quantification of beta-globin target sequences and calculation of the ratio of HCMV copies/beta-globin copies. Serial peripheral blood leukocyte specimens of 17 renal allograft recipients positive in a qualitative nested HCMV PCR were tested using nPCR/TGGE. Thirty healthy blood donors served as negative controls. Positive results were obtained by nPCR/TGGE in nine renal allograft recipients but in none of the healthy blood donors. Five of five patients with an HCMV pp65 antigenaemia and positive for HCMV IgM were positive in nPCR/TGGE. The highest HCMV/beta-globin ratios (10,000 to 8000 copies HCMV/10(6) copies beta-globin) were found in transplant recipients experiencing acute clinically symptomatic HCMV infection. HCMV DNA levels in asymptomatic patients ranged from 900 to 200 copies HCMV/10(6) beta-globin.

Seroprevalence of human polyomavirus 9 and cross-reactivity to African green monkey-derived lymphotropic polyomavirus

Human polyomavirus 9 (HPyV9) was discovered recently in immunocompromised patients and shown to be genetically closely related to B-lymphotropic polyomavirus (LPyV). No serological data are available for HPyV9, but human antibodies against LPyV have been reported previously. To investigate the seroepidemiology of HPyV9 and the sero-cross-reactivity between HPyV9 and LPyV, a capsomer-based IgG ELISA was established using the major capsid protein VP1 of HPyV9 and LPyV. VP1 of an avian polyomavirus was used as control. For HPyV9, a seroprevalence of 47 % was determined in healthy adults and adolescents (n = 328) and 20 % in a group of children (n =101). In both groups, the seroreactivities for LPyV were less frequent and the ELISA titres of LPyV were lower. Of the HPyV9-reactive sera, 47 % reacted also with LPyV, and the titres for both PyVs correlated. Sera from African green monkeys, the natural hosts of LPyV, reacted also with both HPyV9 and LPyV, but here the HPyV9 titres were lower. This potential sero-cross-reactivity between HPyV9 and LPyV was confirmed by competition assays, and it was hypothesized that the reactivity of human sera against LPyV may generally be due to cross-reactivity between HPyV9 and LPyV. The HPyV9 seroprevalence of liver transplant recipients and patients with neurological dysfunctions did not differ from that of age-matched controls, but a significantly higher seroprevalence was determined in renal and haematopoietic stem-cell transplant recipients, indicating that certain immunocompromised patient groups may be at a higher risk for primary infection with or for reactivation of HPyV9.

Heparin increases the infectivity of Human Papillomavirus type 16 independent of cell surface proteoglycans and induces L1 epitope exposure

Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV‐16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV‐16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell‐binding receptors for HPV‐16, heparin‐preincubated virus bound to the extracellular matrix (ECM) via laminin‐332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S‐domains of heparan sulfate (HS) chains of HSPGs, allowed HPV‐16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope‐specific antibody to the viral capsid after heparin binding suggested that initial conformational changes in the HPV‐16 virion occur during infection by interaction with‘heparin‐like’ domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV‐16 infection.

A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla)

Adenoviruses (AdV) broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL), preterminal protein (pTP) and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2 - 10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B). Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses.

Evaluation of Two Enzyme Immunoassays for Detection of Human Rotaviruses in Fecal Specimens

ABSTRACT The two assays evaluated in this study (the Ridascreen rotavirus and the Pathfinder rotavirus) exhibited comparable sensitivities (100%) but highly divergent positive predictive values (93.74 and 57.7%, respectively) when compared on 393 specimens. This difference should be considered when using these tests on collectives with an unknown or low prevalence.

Removal of N-linked Carbohydrates Decreases the Infectivity of Herpes Simplex Virus Type 1

Purified preparations of herpes simplex virus type 1 Angelotti were digested with the exoglycosidases sialidase, beta-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-mannosidase, and with the endoglycosidases Endo-H and Endo-F. It was found that treatment of virions with Endo-F specifically decreased viral infectivity by a factor of 10. This reduction in titre was not associated with any measurable differences in virus adsorption, suggesting a role of N-linked complex type oligosaccharide chains in penetration. In contrast, a reduction in titre observed upon digestion of virions with exoglycosidases could be attributed to a proteolytic contamination in these enzyme preparations. Treatment of virions with Endo-H, demonstrated to be free of proteolytic contamination, did not reduce viral infectivity. Analysis of endoglycosidase-digested virions by monospecific antibodies and immunoblotting revealed a susceptibility of all four major glycoproteins (gC, gB, gE and gD) to Endo-F, but only gB was susceptible to Endo-H treatment. In contrast, of all the exoglycosidases used only sialidase was found to be active towards native viral glycoproteins. Upon analysis of endoglycosidase-digested virions we could not find any evidence for proteolysis, degradation or altered protein composition of viral envelopes. In contrast, vigorous inhibition of glycoprotein glycosylation by tunicamycin led to the formation of physically intact virions almost completely lacking all major glycoproteins. These data show that digestion of intact virions with glycosidases allows an analysis of the functional relevance of carbohydrate residues without any obvious alterations in the virion glycoprotein composition.

Polymerase chain reaction (PCR) from ficoll-purified polymorphonuclear leukocytes for monitoring cytomegalovirus infections in renal allograft recipients: superior sensitivity and similar specificity compared with plasma PCR.

To the Editor—Boivin et al. [1] compared the diagnostic value of quantitative polymerase chain reaction (PCR) using polymorphonuclear leukocytes (PMNL) with qualitative plasma PCR to monitor human immunodeficiency virus–infected individuals at risk for cytomegalovirus (CMV) disease. Symptomatic patients had significantly higher CMV DNA copies in both leukocytes and plasma than asymptomatic individuals, but plasma PCR was far less sensitive than leukocyte PCR due to significantly higher CMV DNA load in leukocytes. Although the specificity and positive predictive value were much lower for a positive leukocyte PCR versus plasma PCR result, this problem was overcome by defining a cutoff value for the CMV DNA load in leukocytes. The authors concluded from their data that quantitative leukocyte PCR was superior to plasma PCR. We would like to comment on three of the issues raised in the discussion. First, Boivin et al. [1] see a discrepancy between their own observations and those reported by others, who were unable to define a reliable cutoff value for leukocyte PCR. However, we previously showed for renal allograft recipients [2] that CMV DNA load in total peripheral blood leukocytes (PBL) of ≥10,000 copies of CMV/10 copies of b-globin DNA (the theoretical average DNA yield from ∼1 mL of whole blood) is highly indicative of clinically manifest CMV infection. This is almost the same as the cutoff of 16,000/mL described by Boivin et al. [1]. Furthermore, we observed similar sensitivity, specificity, and positive and negative predictive values (100%, 94%, 83%, and 100%, respectively). These similarities are all the more remarkable because we investigated a different patient clientele and also used a completely different quantification method based on competitive PCR [3]. Second, Boivin et al. [1] hint that reliable performance of quantitative PCR can be hazardous. In our experience with leukocyte PCR, the DNA extraction procedure is the most crucial step. We regard the parallel quantitation of cellular DNA sequences and CMV genomes as indispensable to assess the efficiency of DNA extraction, since we have observed high interassay variability of total DNA yield from leukocyte samples [3]. Finally, due to the technical demands of quantitative PCR procedures, we find it desirable to improve the diagnostic value of qualitative CMV PCR. Since it is generally accepted that leukocyte PCR is more sensitive and usually becomes positive earlier than plasma PCR, the challenge is to improve the inferior specificity of leukocyte PCR by selective detection of CMV DNA originating from active infection. CMV DNA is found predominantly in PMNL during active infection [4], whereas latently CMV-infected cells belong mostly to the peripheral blood mononuclear cell (PBMC) population [5]. PCR analysis of purified PMNL is therefore the most promising strategy. However, the dextran sedimentation method as used by Boivin et al. [1] and many others for isolating “PMNL” in reality yields leukocyte mixtures containing up to 30% PBMC. This might be a reason for the low specificity of leukocyte PCR. In contrast, using ficoll-metrizoate density centrifugation, which yields ∼95%-pure PMNL and PBMC fractions, we recently found [6] that with leukocyte samples of 35 CMV IgG–positive kidney transplant patients with no laboratory evidence for active CMV infection, only 6% of PMNL but 66% of PBMC fractions were PCR-positive. To assess the diagnostic feasibility of our approach, we performed qualitative PCR from total PBL, ficoll-purified PMNL, and PBMC, and plasma samples from 96 renal allograft recipients to diagnose (1) active CMV infections (n 5 ) as defined by pp65 antigenemia [7] or (2) symptomatic CMV 37 infections ( ) as defined by previously described criteria n 5 19 [8] (table 1). Active as well as symptomatic CMV infections were detected in the PMNL fraction with maximal sensitivity and similar specificity compared with plasma. In contrast, the vast majority of unwanted positive results in leukocytes was attributable to the PBMC. These results suggest that qualitative CMV PCR using ficoll-purified PMNL is a true alternative to plasma PCR and to quantitative leukocyte PCR for monitoring patients at risk for CMV infections.

Prokaryotic expression of the major capsid protein of human cytomegalovirus and antigenic cross-reactions with herpes simplex virus type 1.

The major capsid protein (MCP) of human cytomegalovirus (HCMV) was expressed in three portions as beta-galactosidase fusion proteins, covering about 75% of the open reading frame (ORF). Fusion protein SH 1 contained nucleotides 101 to 1243 of the ORF, fusion protein FS 1 contained nucleotides 1944 to 3089 and fusion protein SS 1 covered nucleotides 2624 to 3793. The recombinant proteins were tested for their immunoreactivity with human sera. Fusion protein FS 1 was found to represent the immunodominant region. The recombinant proteins were used to generate polyvalent rabbit antisera to investigate cross-reactivities with the major capsid protein (VP5) of herpes simplex virus type 1 (HSV-1). A monospecific antiserum raised against the fusion protein close to the N terminus of the MCP, as well as a monoclonal antibody and a monospecific rabbit antiserum directed against the viral MCP, cross-reacted with the VP5 as shown by immunoblotting and immunofluorescence. In order to detect common epitopes of the major capsid proteins of HCMV and HSV-1, the recombinant proteins were conjugated to CNBr-activated Sepharose and taken for purification of MCP antibodies from HCMV and HSV-1 seropositive individuals. Using this affinity chromatography method, cross-reactivity could be observed with HCMV- and HSV-positive human antisera in immunoblot experiments.