Structural and functional analysis of natural capsid variants suggests sialic acid-independent entry of BK polyomavirus.

BK polyomavirus (BKPyV) is an opportunistic pathogen that uses the b-series gangliosides GD1b and GT1b as entry receptors. Here, we characterize the impact of naturally occurring VP1 mutations on ganglioside binding, VP1 protein structure, and virus tropism. Infectious entry of single mutants E73Q and E73A and the triple mutant A72V-E73Q-E82Q (VQQ) remains sialic acid dependent, and all three variants acquire binding to a-series gangliosides, including GD1a. However, the E73A and VQQ variants lose the ability to infect ganglioside-complemented cells, and this correlates with a clear shift of the BC2 loop in the crystal structures of E73A and VQQ. On the other hand, the K69N mutation in the K69N-E82Q variant leads to a steric clash that precludes sialic acid binding. Nevertheless, this mutant retains significant infectivity in 293TT cells, which is not dependent on heparan sulfate proteoglycans, implying that an unknown sialic acid-independent entry receptor for BKPyV exists.

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

The 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.

Quantification of APOBEC3 Mutation Rates Affecting the VP1 Gene of BK Polyomavirus In Vivo.

Mutations in the BK polyomavirus (BKPyV) capsid accumulate in kidney transplant (KTx) recipients with persistent virus replication. They are associated with neutralization escape and appear to arise as a result of cytosine deamination by host cell APOBEC3A/B enzymes. To study the mutagenic processes occurring in patients, we amplified the typing region of the VP1 gene, sequenced the amplicons to a depth of 5000-10,000×, and identified rare mutations, which were fitted to COSMIC mutational signatures. Background mutations were identified in amplicons from plasmids carrying the BKPyV genome and compared to mutations observed in 148 samples from 23 KTx recipients in France and in Vietnam. Three mutational signatures were consistently observed in urine, serum, and kidney biopsy samples, two of which, SBS2 and SBS13, corresponded to APOBEC3A/B activity. In addition, a third signature with no known etiology, SBS89, was detected both in patient samples, and in cells infected in vitro with BKPyV. Quantitatively, APOBEC3A/B mutation rates in urine samples were strongly correlated with urine viral load, and also appeared to vary between individuals. These results confirm that APOBEC3A/B is a major, but not the only, source of BKPyV genome mutations in patients.

Epidemiology and Clinical Impact of Respiratory Coinfections at Diagnosis of Pneumocystis jirovecii Pneumonia.

BACKGROUND: The role of respiratory coinfections at diagnosis of Pneumocystis jirovecii pneumonia (PcP) on clinical impact has been underestimated. METHODS: A retrospective observational study was conducted January 2011 to April 2019 to evaluate respiratory coinfections at diagnosis of PcP patients in 2 tertiary care hospitals. Coinfection was defined by identification of pathogens from P. jirovecii-positive samples. RESULTS: Of 7882 respiratory samples tested for P. jirovecii during the 8-year study, 328 patients with diagnosis of PcP were included. Mean age was 56.7 (SD 14.9) years, 193 (58.8%) were male, 74 (22.6%) had positive HIV serology, 125 (38.1%) had viral coinfection, 76 (23.2%) bacterial coinfection, and 90-day mortality was 25.3%. In the overall population, 90-day mortality was independently associated with solid tumor underlying disease (odds ratio [OR], 11.8; 95% confidence interval [CI], 1.90-78.0; P = .008), sepsis-related organ failure assessment score (SOFA) at admission (OR, 1.62; 95% CI, 1.34-2.05; P< .001), and cytomegalovirus (CMV) respiratory coinfection (OR, 3.44; 95% CI, 1.24-2.90; P = .02). Among HIV-negative patients, respiratory CMV coinfection was associated with worse prognosis, especially when treated with adjunctive corticosteroid therapy. CONCLUSIONS: Respiratory CMV coinfection at PcP diagnosis was independently associated with increased 90-day mortality, specifically in HIV-negative patients.

Non-permissive human conventional CD1c+ dendritic cells enable trans-infection of human primary renal tubular epithelial cells and protect BK polyomavirus from neutralization.

The BK polyomavirus (BKPyV) is a ubiquitous human virus that persists in the renourinary epithelium. Immunosuppression can lead to BKPyV reactivation in the first year post-transplantation in kidney transplant recipients (KTRs) and hematopoietic stem cell transplant recipients. In KTRs, persistent DNAemia has been correlated to the occurrence of polyomavirus-associated nephropathy (PVAN) that can lead to graft loss if not properly controlled. Based on recent observations that conventional dendritic cells (cDCs) specifically infiltrate PVAN lesions, we hypothesized that those cells could play a role in BKPyV infection. We first demonstrated that monocyte-derived dendritic cells (MDDCs), an in vitro model for mDCs, captured BKPyV particles through an unconventional GRAF-1 endocytic pathway. Neither BKPyV particles nor BKPyV-infected cells were shown to activate MDDCs. Endocytosed virions were efficiently transmitted to permissive cells and protected from the antibody-mediated neutralization. Finally, we demonstrated that freshly isolated CD1c+ mDCs from the blood and kidney parenchyma behaved similarly to MDDCs thus extending our results to cells of clinical relevance. This study sheds light on a potential unprecedented CD1c+ mDC involvement in the BKPyV infection as a promoter of viral spreading.

[Inflammatory cardiomyopathies: Diagnosis, correlations with imaging, interest of myocardial biopsy and place of molecular biology]. / Cardiomyopathies inflammatoires : diagnostic, corrélations avec l'imagerie, intérêt de la biopsie myocardique et place de la biologie moléculaire.

Inflammatory cardiomyopathies, also known as "myocarditis" are inflammatory pathologies affecting the myocardium and characterized by vast etiological and clinical heterogeneity. They can be asymptomatic, particularly in viral forms, or be responsible for sudden death, particularly in subjects under 35 years olds. Due to insufficient sensitivity and specificity of imaging and biology, the gold standard is histopathological and is performed on an endomyocardial biopsy or on explanted heart samples in a transplant context. Their classification has considerably evolved and is now based on the identification of a predominant cell pattern such as lymphocytic, neutrophilic or eosinophilic polynuclear, giant cell or granulomatous myocarditis. These different patterns will guide the etiological diagnosis, prognosis and the therapies to be implemented. Due to the importance of viral etiologies, this morphological analysis must be complemented by a virological analysis based on PCR with viral load quantification. In addition, some authors have been able to demonstrate the occurrence of myocarditis in patients with arrhythmogenic cardiomyopathy of genetic origin. The aim of this chapter is to review the current state of knowledge on inflammatory cardiomyopathies and their management.

Persistent BK Polyomavirus Viruria is Associated with Accumulation of VP1 Mutations and Neutralization Escape.

To investigate the relationship between neutralization escape and persistent high-level BK polyomavirus replication after kidney transplant (KTx), VP1 sequences were determined by Sanger and next-generation sequencing in longitudinal samples from KTx recipients with persistent high-level viruria (non-controllers) compared to patients who suppressed viruria (controllers). The infectivity and neutralization resistance of representative VP1 mutants were investigated using pseudotype viruses. In all patients, the virus population was initially dominated by wild-type VP1 sequences, then non-synonymous VP1 mutations accumulated over time in non-controllers. BC-loop mutations resulted in reduced infectivity in 293TT cells and conferred neutralization escape from cognate serum in five out of six non-controller patients studied. When taken as a group, non-controller sera were not more susceptible to neutralization escape than controller sera, so serological profiling cannot predict subsequent control of virus replication. However, at an individual level, in three non-controller patients the VP1 variants that emerged exploited specific "holes" in the patient's humoral response. Persistent high-level BK polyomavirus replication in KTx recipients is therefore associated with the accumulation of VP1 mutations that can confer resistance to neutralization, implying that future BKPyV therapies involving IVIG or monoclonal antibodies may be more effective when used as preventive or pre-emptive, rather than curative, strategies.

Intra-patient viral evolution in polyomavirus-related diseases.

Human 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'.

Fine Mapping the Interaction Between Dendritic Cell-Specific Intercellular Adhesion Molecule (ICAM)-3-Grabbing Nonintegrin and the Cytomegalovirus Envelope Glycoprotein B.

Background: Human cytomegalovirus (HCMV) is a leading cause of virally induced congenital disorders and morbidities in immunocompromised individuals, ie, transplant, cancer, or acquired immune deficiency syndrome patients. Human cytomegalovirus infects virtually all cell types through the envelope glycoprotein complex gH/gL/gO with or without a contribution of the pentameric gH/gL/pUL128L. Together with gH/gL, the HCMV envelope glycoprotein B (gB) contributes to the viral fusion machinery. Methods: We previously showed that gB is a ligand for the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) contributing to HCMV attachment to and infection of DC-SIGN-expressing cells. However, the features of the DC-SIGN/gB interaction remain unclear. To address this point, the role of glycans on gB and the consequences of mutagenesis and antibody-mediated blockades on both partners were examined in this study. Results: We identified DC-SIGN amino acid residues involved in this interaction through an extensive mutagenesis study. We also showed the importance of high-mannose N-glycans decorating the asparagine residue at position 208, demonstrating that the antigenic domain 5 on gB is involved in the interaction with DC-SIGN. Finally, antibody-mediated blockades allowed us to identify DC-SIGN as a major HCMV attachment receptor on monocyte-derived dendritic cells. Conclusions: Taken together, these results have permitted us to fine-map the interaction between DC-SIGN and HCMV gB.

Impact of stem cell graft on early viral infections and immune reconstitution after allogeneic transplantation in adults.

BACKGROUND: Viral infections are well-known complications after allogeneic stem cell transplant (allo-SCT). OBJECTIVES: We compared prospectively incidences of DNAemia and active infections (AI) for five opportunistic viruses (Human Herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), BK polyomavirus (BKPyV), Cytomegalovirus (CMV) and Adenovirus (ADV)) and kinetics of immune reconstitution (IR) in adults receiving either double umbilical cord blood (dUCB group) or unrelated peripheral blood stem cell (uPBSC group) allo-SCT after a reduced-intensity conditioning (RIC) regimen. STUDY DESIGN: Whole blood samples were collected at transplant, every 15days during the first 3 months and at 4, 5 and 6 months post-transplant. RESULTS: Sixty-five patients were enrolled (uPBSC n=34; dUCB n=31). Incidences of HHV-6 and BKPyV DNAemia were significantly higher for dUCB (97% vs 23.5% and 58% vs 32%, respectively) while EBV DNAemia was more frequently detected in uPBSC (71% vs 26%). The incidence of CMV DNAemia was similar between both groups. ADV AI developed only in dUCB. HHV-6 AI were also higher in dUCB (84% vs 21%). In multivariate analysis, dUCB graft was the only independent factor associated with HHV-6 DNAemia (OR: 19.0; 95%CI: 5.2-69.1; p<0.0001) while EBV DNAemia were significantly associated with uPBSC (OR: 29.9; 95%CI: 5.68-158; p <0.0001). dUCB graft was also the only factor associated with HHV-6 AI. Finally, higher counts and faster recoveries of B lymphocytes (p<0.0001) and monocytes (p=0.0007) were observed in the dUCB group. CONCLUSION: We demonstrate a strong correlation between sources of graft and patterns of viral DNAemia and AI and IR after RIC allo-SCT.

MICA Mutant A5.1 Influences BK Polyomavirus Reactivation and Associated Nephropathy After Kidney Transplantation.

BACKGROUND: BK polyomavirus (BKPyV) frequently reactivates in kidney transplant recipients during immunosuppressive therapy and triggers BKPyV-associated nephropathy and graft rejection. Determining effective risk factors for BKPyV reactivation is required to achieve efficient prevention. METHODS: This study investigated the role of major histocompatibility complex (MHC) class I-related chain A (MICA) in BKPyV reactivation in a cohort of 144 transplant donor/recipient pairs, including recipients with no reactivation (controllers) and those with mild (virurics) or severe (viremics) BKPyV reactivation after graft receipt. RESULTS: We show that, in the kidney, MICA is predominantly expressed in tubule epithelial cells, the natural targets of BKPyV, questioning a role for MICA in the immune control of BKPyV infection. Focusing on MICA genotype, we found a lower incidence of BKPyV reactivation in recipients of a renal graft from a donor carrying the MICA A5.1 mutant, which encodes a truncated nonconventional MICA. We established that a mismatch for MICA A5.1 between transplant donor and recipient is critical for BKPyV reactivation and BKPyV-associated nephropathy. Functionally, we found that a low prevalence of BKPyV reactivation was associated with elevated anti-MICA sensitization and reduced plasma level of soluble MICA in recipients, 2 potential effector mechanisms. DISCUSSIONS: These findings identify the MHC-related MICA as an immunogenetic factor that may functionally influence anti-BKPyV immune responses and infection outcomes.

BK polyomavirus: virus-cell interactions, host immune response, and viral pathogenesis.

The BK polyomavirus (BKPyV) is one of the main human polyomaviruses. After primary infection, it establishes a persistent infection, and acts as an opportunistic pathogen, innocuous in immunocompetent hosts, but causing potentially serious pathology in the context of immunosuppression, in particular in kidney and hematopoietic stem cell graft recipients. Much progress has been made in recent years in the description of virus-cell interactions, but many aspects of viral physiopathology remain mysterious, principally due to the asymptomatic nature of infection in immunocompetent individuals and the lack of an animal model. The characteristics of the antiviral immune response are beginning to become more clearly understood, particularly in kidney transplant patients. Work in these areas is important in order to identify patients at high risk of developing a severe infection. Indeed, in the absence of an effective antiviral therapy few therapeutic options are available, and patient management remains based on modulation of immunosuppressive therapy.

Long-lasting HHV-6 reactivation in long-term adult survivors after double umbilical cord blood allogeneic stem cell transplantation.

Higher incidence of human herpesvirus 6 (HHV-6) infection has been documented after umbilical cord blood allo-transplant in adults. Here we demonstrate that HHV-6 reactivation persists for a very long time in half of the patients after this type of graft. Long-term immune reconstitution does not explain this event, which remains to be explained.

Quantification of two viral transcripts by real time PCR to investigate human herpesvirus type 6 active infection.

BACKGROUND: Human herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types. OBJECTIVES: To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting. STUDY-DESIGN: Two one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients. RESULTS: Analytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000 copies/ml of blood. CONCLUSION: The new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection.

Resistance pattern of cytomegalovirus (CMV) after oral valganciclovir therapy in transplant recipients at high-risk for CMV infection.

In transplant recipients, cytomegalovirus (CMV) resistance to antivirals causes an increasing problem. Here we report the clinical, therapeutic, and virological characteristics of 11 cases of CMV resistance among transplant recipients at high-risk for CMV infection and receiving valganciclovir as a prophylactic, preemptive or maintenance therapy. Active CMV infection was monitored by viral DNA quantification in whole blood, and CMV resistance was assessed by UL97 and UL54 viral gene sequencing. For 10 patients, ganciclovir resistance detected after valganciclovir therapy was associated with one mutation within UL97 phosphotransferase located at codons 460 and 592-603, which constitutes a similar pattern of resistance to what has been reported previously in AIDS patients treated with valganciclovir. For the last patient, two mutations in UL97 and UL54 genes were identified. The start of valganciclovir maintenance treatment after an intravenous curative treatment while CMV DNA is still detectable in peripheral blood might represent a risk factor for the emergence of CMV resistance. The possible emergence of CMV resistance in transplant recipients at high-risk for CMV infection who receive valganciclovir therapy should be taken into account. Among those patients, CMV infection has to be closely monitored in order to detect promptly the emergence of drug-resistance.

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre-emptive therapy.

Two quantitative duplex real-time PCR assays were developed for co-amplification of human albumin and cytomegalovirus (CMV) or Epstein Barr virus (EBV) genes after automated extraction on whole blood, and compared two units for expressing viral DNA loads (copies per ml of blood or per 10(6) peripheral blood leukocytes (PBLs)) on 1,138 positive samples. Both PCRs were characterized by high sensitivity, reproducibility, and linear range. Automated extraction by a MagNA Pure LC Instrument was shown to be more efficient when peripheral blood cell count was inferior to 5 x 10(9) PBLs/L. Albumin co-amplification allows the detection of PCR inhibitors and normalization of viral load according to the number of cells calculated in the sample. The two ways of expressing viral load results were highly correlated, but quantitative differences varied in relation to variations of blood cell count. As these two viruses are highly cell associated, viral loads can be underestimated in patients with leucopenia. In the setting of pre-emptive strategies during CMV infection, the units in which results are expressed can influence clinical management, as illustrated in this article.