Quantitation of BK Virus DNA for Diagnosis of BK Virus-Associated Nephropathy in Renal Transplant Recipients

Quantitative measurement of BK virus DNA (Q-BKDNA) has been used for the early diagnosis and monitoring of BK virus-associated nephropathy (BKVAN). This study was designed to determine the BKDNA cutoff for the diagnosis of BKVAN. Between June 2005 and February 2007, 64 renal transplant recipients taken renal biopsies due to renal impairment submitted plasma and urine for Q-BKDNA. Eight BKVAN patients (12.5%) had median viral loads of 6.0 log10 copies/mL in plasma and 7.3 log10 copies/mL in urine. Among 56 non-BKVAN patients, 45 were negative for Q-BKDNA; 4 were positive in plasma with a median viral load of 4.8 log10 copies/mL, and 10 were positive in urine with a median viral load of 4.8 log10 copies/mL. Receiver operating characteristic curve analysis showed that a cutoff of 4.5 log10 copies/mL in plasma and a cutoff of 5.9 log10 copies/mL in urine had a sensitivity of 100% and a specificity of 96.4%, respectively. A combined cutoffs of 4 log10 copies/mL in plasma and 6 log10 copies/mL in urine had better performance with a sensitivity of 100% and a specificity of 98.2% than each cutoff of urine or plasma. Q-BKDNA with the combined cutoffs could reliably diagnose BKVAN in renal transplant recipients

Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a "Trojan horse" to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection

Human cytomegalovirus infection of langerhans-type dendritic cells does not require the presence of the gH/gL/UL128-131A complex and is blocked after nuclear deposition of viral genomes in immature cells

Human cytomegalovirus (CMV) enters its host via the oral and genital mucosae. Langerhans-type dendritic cells (LC) are the most abundant innate immune cells at these sites, where they constitute a first line of defense against a variety of pathogens. We previously showed that immature LC (iLC) are remarkably resistant to CMV infection, while mature LC (mLC) are more permissive, particularly when exposed to clinical-strain-like strains of CMV, which display a pentameric complex consisting of the viral glycoproteins gH, gL, UL128, UL130, and UL131A on their envelope. This complex was recently shown to be required for the infection of immature monocyte-derived dendritic cells. We thus sought to establish if the presence of this complex is also necessary for virion penetration of LC and if defects in entry might be the source of iLC resistance to CMV. Here we report that the efficiency of LC infection is reduced, but not completely abolished, in the absence of the pentameric complex. While virion penetration and nuclear deposition of viral genomes are not impaired in iLC, the transcription of the viral immediate early genes UL122 and UL123 and of the delayed early gene UL50 is substantially lower than that in mLC. Together, these data show that the UL128, UL130, and UL131A proteins are dispensable for CMV entry into LC and that progression of the viral cycle in iLC is restricted at the step of viral gene expression

Développement d'une PCR en temps réel pour la quantification du BK virus (application au suivi d'une cohorte de transplantés rénaux)

Le polyomavirus humain BK, virus ubiquitaire dont la séroprévalence est élevée, ne s'exprime cliniquement que dans un contexte d'immunosuppression. L'incidence des néphropathies à BK virus en transplantation rénale a augmenté depuis le milieu des années 90. Nous avons mis au point une PCR en temps réel pour la quantification de l'ADN viral. Disposant ainsi d'un outil sensible, spécifique et reproductible, nous avons suivi prospectivement une cohorte de patients transplantés depuis Décembre 2001. Nos résultats montrent une incidence élevée de l'ADNurie et de l'ADNémie BK virus. Aucun cas de néphropathie associée n'a été diagnostiqué au cours des 12 premiers mois. Nos travaux suggèrent le rôle favorisant du traitement immunosuppresseur sur la réactivation du BK virus, et soulignent l'intérêt de la mesure de la charge virale plasmatique pour le suivi des néphropathies à BK virus et l'adaptation du traitement.NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Intra-patient viral evolution in polyomavirus-related diseases

International audienceHuman polyomaviruses show relatively little genetic polymorphism between isolates, indicating that these viruses are genetically stable between hosts. However, it has become increasingly clear that intra-host molecular evolution is a feature of some polyomavirus (PyV) infections in humans. Mutations inducing premature stop codons in the early region of the integrated Merkel cell PyV genome lead to the expression of a truncated form of the large tumour (LT) antigen that is critical for the transformation of Merkel cell carcinoma (MCC) cells. Non-coding control region (NCCR) rearrangements and point mutations in virion protein (VP) 1 have been described in both JCPyV and BKPyV infections. In the context of JCPyV infection, molecular evolution at both these loci allows the virus to replicate effectively in the central nervous system, thereby leading to the development of progressive multifocal leukoencephalopathy (PML). In BKPyV infection, NCCR rearrangements have been linked to higher rates of virus replication in the kidney, and are proposed to play a direct causal role in the development of PyV-associated nephropathy. In all three of these infections, therefore, intra-host viral evolution appears to be an essential component of the disease process. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'

Le cytomégalovirus humain (moyens d'étude de la multiplication)

Le cytomégalovirus humain (CMVH), membre de la famille des Herpesviridae, est un virus ubiquitaire dont le pouvoir pathogène lors de l'infection active est étroitement associé à la qualité de la réponse immunitaire. La grande taille de son génome lui permet de coder pour de nombreuses protéines qui peuvent moduler la réponse immune. Les génomes des souches cliniques de CMVH présentent une homologie de séquence de l'ordre de 90, mais il existe des zones de polymorphisme au sein de régions du génome codant notamment pour des protéines impliquées dans le tropisme cellulaire des différentes souches de CMVH ou dans les mécanismes d'échappement à la réponse immunitaire. Nous avons étudié le polymorphisme des gènes IE1, UL55 et UL22A dans une cohorte de patients. L'étude phylogénique de la séquence complète du gène UL22A, qui code une protéine glycosylée et sécrétée capable de se lier spécifiquement à la chimiokine CCL5, a permis de différencier trois groupes génomiques dont la répartition n'est pas identique entre les différentes populations de patients (VIH, allogreffés ou immunocompétents). Des outils de mesure de la réplication (PCR duplex CMV/albumine) et de la transcription virale (RT PCR ffil, MCP et UL22A) basés sur des méthodes d'amplification génique par technologie temps réel ont été développés pour suivre la multiplication virale in vitro et in vivo afin de comparer le comportement des différentes souches virales entre elles. Une technique de détection d'un antigène viral très précoce par cytométrie en flux a également été développée. Ces méthodes ont été appliquées au suivi de la réplication de deux souches de CMVH dénommées TB40/E et VHL/E, caractérisées par un tropisme endothélial conservé, sur des fibroblastes et des cellules dendritiques. Les cellules dendritiques sont un élément clé de la réponse immunitaire dirigée contre le CMVH, mais sont également une des cibles du virus. En étudiant le niveau d'expression de molécules de co-stimulation (B7.1 et B7.2), de maturation (CD83) et des molécules du CMH, nous avons montré que les modifications phénotypiques de cellules dendritiques immatures secondaires à l'exposition au virus varient en fonction de l'inoculum viral. Les conséquences phénotypiques de l'infection de polynucléaires neutrophiles par des souches cliniques de CMVH ont également été étudiées. Les polynucléaires neutrophiles sont un des vecteurs de dissémination virale au cours de l'infection active et sont capables de capter et de transmettre le virus par interactions directes de cellule à cellule. Nous avons observé des modifications du niveau d'expression de certaines intégrines leucocytaires (CDllb, CDllc et CD18) impliquées dans les mécanismes d'adhésion des polynucléaires neutrophiles. L'ensemble de ces résultats contribue à l'exploration des différents facteurs de pathogénicité au cours de l'infection à CMVH.aHuman cytomegalovirus is an ubiquitous member of the Herpesviridae family. Its pathogenicity is highly correlated with the efficacy of the immune response during active infection. HCMV has the largest genome among the herpesviruses, and it encodes a large number of proteins involved in the modulation of immune responses. There is a high percentage of sequence homology between the clinical strains, but some regions of the genome involved in cell tropism or immune modulation mechanisms are characterised by genetic polymorphism. Genetic polymorphism of three viral genes (IE1, UL22A and UL55) was studied in a large patient population. Phylogenetie analysis of the UL22A gene, which encodes a secreted glycoprotein that binds to the CCL5 chemokine, differentiated three genetic clusters. The distribution of these clusters was not equal when comparing three patient groups: patients with HIV, allograft transplant recipients and immunocompetent subjects. Methods for the monitoring of HCMV replication (duplex PCR CMV/albumine) and transcription (IE1, UL22A and MCP RT PCR), based on real time technology, were developed in order to compare the behaviour of HCMV strains both in vivo and in vitro. We also developed a flow cytometry method for intracellular detection of viral antigens after virus exposure of different cell types. The replication kinetics of two HCMV strains (TB40E and VHLE), characterised by their endothelial tropism, was studied in infected fibroblasts and dendritic cells. Dendritic cells are a key element of the immune response directed against HCMV during the early stages of active infection. Expression of the co-stimulatory molecules (B7.1 and B7.2), the maturation marker (CD83) and MHC class I molecules was shown to be modified after infection with either TB40E or VHLE strains and we observed variations in these modifications depending on the initial viral inoculum. We also studied phenotypic modifications on polymorphonuelear cells infected with clinical strains. Polymorphonuclear cells are involved in the propagation of the virus during active infection. Viral particle uptake by these cells is mediated by direct interaction with other infected cells and involves adhesion mechanisms. We observed alterations in the expression of certain adhesion molecules (CD lib, CD lie and CD 18) subsequent to polymorphonuelear cell infection. All together, these results contribute to the exploration of factors that could influence viral pathogenesis.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Caractérisation des interactions entre les cellules dendritiques myéloïdes humaines et le BKPyV

National audienceLe polyomavirus BK (ou BKPyV) est un virus ubiquitaire infectant de manière asymptomatique 80 % de la population pendant l’enfance. Suite à la primo-infection, il entre en phase de réplication à bas bruit dans les epithelia réno-urinaires. Suite à une transplantation, la prise d’un traitement immunosuppresseur diminue les réponses immunitaires et donc le contrôle des infections virales notamment. Dans ce contexte, le BKPyV se comporte en pathogène opportuniste et se réplique activement dans le rein. Cette infection active peut en outre induire le développement d’une néphropathie évolutive, la néphropathie associée aux polyomavirus ou PVAN, caractérisée par une virémie et pouvant conduire au rejet du greffon. La gravité de cette atteinte rénale a été corrélée à l’établissement de cette virémie, dont la cause reste mal connue. Ainsi, le BKPyV est reconnu comme une cause infectieuse majeure de rejet de greffe. Basé sur des propriétés des cellules dendritiques (DC) aujourd’hui bien décrites, telles que la capacité à protéger des virus en surface et à les transmettre à d’autres cellules, et l’observation de DC à proximité immédiate des sites de réactivation du virus dans le rein normal, notre hypothèse est que les DC sont capables de capter le BKPyV et de le transporter avec elles dans l’organisme, favorisant l’établissement de la virémie. Notre groupe a ainsi pu démontrer par cytométrie de flux et par imagerie confocale, que des particules virales infectieuses, mais également des pseudo-particules (VLP), peuvent être capturées spécifiquement par des cellules dendritiques myéloïdes (mDC) in vitro. En outre, nous avons également démontré que la protéine VP1, composant majeur de la capside, était incapable d’activer les mDC. Enfin, nous avons montré que le virus capturé par les mDC peut être transmis à des cellules épithéliales tubulaires rénales très susceptibles à l’infection BKPyV. En conclusion, nous pouvons dire que cette interaction entre des particules infectieuses de BKPyV avec notre modèle de mDC est décrite pour la première fois. Elle doit être confirmée dans d’autres modèles et approfondie. Nous envisageons en collaboration avec les néphrologues nantais d’investiguer le rôle de ces cellules dans les greffons de reins humains après transplantation

Virus-Associated Nephropathies: A Narrative Review

International audienceWhile most viral infections cause mild symptoms and a spontaneous favorable resolution, some can lead to severe or protracted manifestations, specifically in immunocompromised hosts. Kidney injuries related to viral infections may have multiple causes related to the infection severity, drug toxicity or direct or indirect viral-associated nephropathy. We review here the described virus-associated nephropathies in order to guide diagnosis strategies and treatments in cases of acute kidney injury (AKI) occurring concomitantly with a viral infection. The occurrence of virusassociated nephropathy depends on multiple factors: the local epidemiology of the virus, its ability to infect renal cells and the patient's underlying immune response, which varies with the state of immunosuppression. Clear comprehension of pathophysiological mechanisms associated with a summary of described direct and indirect injuries should help physicians to diagnose and treat viral associated nephropathies

Distinctive phenotype for HLA-E- versus HLA-A2-restricted memory CD8 αβT cells in the course of HCMV infection discloses features shared with NKG2C+CD57+NK and δ2-γδT cell subsets

International audienceThe human cytomegalovirus (HCMV) triggers both innate and adaptive immune responses, including protective CD8+ αβT cells (CD8T) that contributes to the control of the infection. In addition to CD8T restricted by classical HLA class Ia molecules, HCMV also triggers CD8T recognizing peptides from the HCMV UL40 leader peptide and restricted by HLA-E molecules (HLA-EUL40 CD8T). This study investigated the frequency, phenotype and functions of HLA-EUL40 CD8T in comparison to the immunodominant HLA-A2pp65 CD8T upon acute (primary or secondary infection) or chronic infection in kidney transplant recipients (KTR) and in seropositive (HCMV+) healthy volunteer (HV) hosts. The frequency of hosts with detected HLA-EUL40 CD8T was similar after a primary infection (24%) and during viral latency in HCMV+ HV (26%) and equal to the frequency of HLA-A2pp65 CD8T cells in both conditions (29%). Both CD8T subsets vary from 0.1% to >30% of total circulating CD8T according to the host. Both HLA-EUL40 and HLA-A2pp65 CD8T display a phenotype specific of CD8+ TEMRA (CD45RA+/CCR7-) but HLA-EUL40 CD8T express distinctive level for CD3, CD8 and CD45RA. Tim3, Lag-3, 4-1BB, and to a lesser extend 2B4 are hallmarks for T cell priming post-primary infection while KLRG1 and Tigit are markers for restimulated and long lived HCMV-specific CD8T responses. These cell markers are equally expressed on HLA-EUL40 and HLA-A2pp65 CD8T. In contrast, CD56 and PD-1 are cell markers discriminating memory HLA-E- from HLA-A2-restricted CD8T. Long lived HLA-EUL40 display higher proliferation rate compared to HLA-A2pp65 CD8T consistent with elevated CD57 expression. Finally, a comparative immunoprofiling indicated that HLA-EUL40 CD8T, divergent from HLA-A2pp65 CD8T, share the expression of CD56, CD57, NKG2C, CD158 and the lack of PD-1 with NKG2C+CD57+ NK and δ2-γδT cells induced in response to HCMV and thus defines a common immunopattern for these subsets