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

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

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

Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses

International audiencePrimary human cytomegalovirus (HCMV) infection usually goes unnoticed, causing mild or no symptoms in immunocompetent individuals. However, some rare severe clinical cases have been reported without investigation of host immune responses or viral virulence. In the present study, we investigate for the first time phenotypic and functional features, together with gene expression profiles in immunocompetent adults experiencing a severe primary HCMV infection. Twenty primary HCMV-infected patients (PHIP) were enrolled, as well as 26 HCMV-seronegative and 39 HCMV-seropositive healthy controls. PHIP had extensive lymphocytosis marked by massive expansion of natural killer (NK) and T cell compartments. Interestingly, PHIP mounted efficient innate and adaptive immune responses with a deep HCMV imprint, revealed mainly by the expansion of NKG2C+ NK cells, CD16+ Vδ2(-) γδ T cells, and conventional HCMV-specific CD8+ T cells. The main effector lymphocytes were activated and displayed an early immune phenotype that developed toward a more mature differentiated status. We suggest that both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage observed in PHIP. Taken together, these findings bring new insights into the comprehensive understanding of immune mechanisms involved during primary HCMV infection in immunocompetent individuals.IMPORTANCE HCMV-specific immune responses have been extensively documented in immunocompromised patients and during in utero acquisition. While it usually goes unnoticed, some rare severe clinical cases of primary HCMV infection have been reported in immunocompetent patients. However, host immune responses or HCMV virulence in these patients has not so far been investigated. In the present study, we show massive expansion of NK and T cell compartments during the symptomatic stage of acute HCMV infection. The patients mounted efficient innate and adaptive immune responses with a deep HCMV imprint. The massive lymphocytosis could be the result of nonadapted or uncontrolled immune responses limiting the effectiveness of the specific responses mounted. Both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage. Furthermore, we cannot exclude a delayed immune response caused by immune escape established by HCMV strains

Association of Dynamics of Anellovirus Loads With Hospital-Acquired Pneumonia in Patients With Brain Injury During the Intensive Care Unit Stay

International audienceBackground : Critical illness induces immune disorders associated with an increased risk of hospital-acquired pneumonia (HAP) and acute respiratory distress syndrome (ARDS). Torque teno virus (TTV), from the Anelloviridae family, is proposed as a biomarker to measure the level of immunosuppression. Our objective was to describe the kinetics of TTV DNA loads and their association with critical illness–related complications. Methods : We performed a longitudinal study in 115 patients with brain injury from a prospective cohort, collected endotracheal and blood samples at 3 successive time points after admission in the intensive care unit (ICU) (T1, 0–4 days post ICU admission; T2, 5–10; T3, 11–18), and measured viral DNA loads using the TTV R-GENE kit (BioMérieux) and a pan-Anelloviridae in-house quantitative real-time polymerase chain reaction. Results : TTV DNA was detected in the blood of 69%, 71%, and 64% of patients with brain injury at T1, T2, and T3, respectively. Time-associated variations of TTV and anellovirus DNA loads were observed. Using a linear mixed-effects model, we found that HAP and ARDS were associated with lower blood anellovirus DNA loads. Conclusions : Our results show that HAP or ARDS in patients who are critically ill is associated with changes in anellovirus DNA loads and should be evaluated further as a biomarker of immune disorders leading to these complications

A cluster of broadly neutralizing IgG against BK polyomavirus in a repertoire dominated by IgM

International audienceThe BK polyomavirus (BKPyV) is an opportunistic pathogen, which is only pathogenic in immunosuppressed individuals, such as kidney transplant recipients, in whom BKPyV can cause significant morbidity. To identify broadly neutralizing antibodies against this virus, we used fluorescence-labeled BKPyV virus-like particles to sort BKPyV-specific B cells from the PBMC of KTx recipients, then single-cell RNAseq to obtain paired heavy- and light-chain antibody sequences from 2,106 sorted B cells. The BKPyV-specific repertoire was highly diverse in terms of both V-gene usage and clonotype diversity and included most of the IgM B cells, including many with extensive somatic hypermutation. In two patients where sufficient data were available, IgM B cells in the BKPyV-specific dataset had significant differences in V-gene usage compared with IgG B cells from the same patient. CDR3 sequence–based clustering allowed us to identify and characterize three broadly neutralizing “41F17-like” clonotypes that were predominantly IgG, suggesting that some specific BKPyV capsid epitopes are preferentially targeted by IgG