Caractérisation du virus BK et rôle des vésicules extracellulaires sur sa pathogénicité

During the last years, development of new strong immunosuppressive treatment protocols used in renal transplantation has allowed a better prevention of allograft rejection. However, the induced immunodeficiency leads to the reactivation of multiple viral agents, causative of pathologies with potentially serious consequences such as transplant rejection. The BK virus (BKPyV) is one of these opportunistic viruses. It replicates among one in two transplant patients during the first year post-transplant while more than 80% of the general population is asymptomatic carrier. Unfortunately, its mechanisms of persistence and immune evasion remain misunderstood today. We purified the virus produced in cell culture on a iodixanol gradient and we found that infected cells released two infectious populations of different densities. One of them co-sedimented with extracellular vesicles (EV). This population was less sensitive to protease digestion and its treatment by chloroform leads to the the disappearance of the low-density BKPyV population whereas it increases the high-density population. Electron microscopy allowed us to confirm that EV could transport about ten virions. We also found that the particles included in the EV (eBKPyV), unlike naked particles, are no longer able to agglutinate red blood cells and don’t need sialylated glycan groups to infect cells. The existence of two BKPyV forms could allow diversification of its infection modes. Furthermore, we also observed that naked virus and eBKPyV were both sensitive to neutralization by antibodies from an infected patient and by polyvalent commercial immunoglobulins, with no significant difference, and that this neutralization occured after endocytosis. The presence of BKPyV in extracellular vesicles, a phenomenon called "transmission en bloc", could have a critical incidence during primary infection, persistence but also reactivation of the virus and could be a new target in the development of new therapiesAu cours de ces dernières années, le développement de nouveaux protocoles de traitements immunosuppresseurs de plus en plus puissants en transplantation rénale a permis une meilleure prévention des rejets d'allogreffes. Cependant, l'immunodéficience induite conduit à la réactivation de multiples agents viraux responsables de pathologies aux conséquences graves comme un rejet du greffon. Le virus BK (BKPyV) est un de ces virus opportunistes. Il se réplique chez un patient greffé sur deux au cours de la première année post-greffe tandi’s que plus de 80% de la population générale est porteuse asymptomatique. Malheureusement, ses mécanismes de persistance et d'échappement au système immunitaire demeurent encore incompris. En purifiant le virus, nous avons constaté que les cellules infectées relarguaient deux populations infectieuses de densités différentes, dont l'une co-sédimente avec les vésicules extracellulaires (VE). Cette population est par ailleurs moins sensible à l'action de protéases et, sous l'action du chloroforme, retrouve une densité similaire à celle des virions nus. La microscopie électronique nous a permis de confirmer que des VE pouvaient transporter une dizaine de virions. Nous avons alors constaté que les particules comprises dans les VE (eBKPyV), contrairement aux particules nues, ne sont plus hémagglutinantes et n'ont pas besoin des groupements de glycanes sialylés pour entrer dans les cellules. Cette dualité de formes permet donc une diversification des modes d'entrée. Nous avons aussi observé que virus nu et eBKPyV étaient sensibles à la neutralisation par des anticorps de patient infecté et par des immunoglobulines polyvalentes et que cette neutralisation avait lieu après endocytose. La présence du BKPyV au sein de VE, phénomène portant le nom de "transmission en bloc", pourrait finalement jouer un rôle critique au cours de l'infection primaire, de la persistance, mais aussi de la réactivation du virus et offrir une nouvelle cible dans le développement de nouvelles thérapie

Characterization of the BK virus and role of the extracellular vesicles on its pathogenicity

Au cours de ces dernières années, le développement de nouveaux protocoles de traitements immunosuppresseurs de plus en plus puissants en transplantation rénale a permis une meilleure prévention des rejets d'allogreffes. Cependant, l'immunodéficience induite conduit à la réactivation de multiples agents viraux responsables de pathologies aux conséquences graves comme un rejet du greffon. Le virus BK (BKPyV) est un de ces virus opportunistes. Il se réplique chez un patient greffé sur deux au cours de la première année post-greffe tandi’s que plus de 80% de la population générale est porteuse asymptomatique. Malheureusement, ses mécanismes de persistance et d'échappement au système immunitaire demeurent encore incompris. En purifiant le virus, nous avons constaté que les cellules infectées relarguaient deux populations infectieuses de densités différentes, dont l'une co-sédimente avec les vésicules extracellulaires (VE). Cette population est par ailleurs moins sensible à l'action de protéases et, sous l'action du chloroforme, retrouve une densité similaire à celle des virions nus. La microscopie électronique nous a permis de confirmer que des VE pouvaient transporter une dizaine de virions. Nous avons alors constaté que les particules comprises dans les VE (eBKPyV), contrairement aux particules nues, ne sont plus hémagglutinantes et n'ont pas besoin des groupements de glycanes sialylés pour entrer dans les cellules. Cette dualité de formes permet donc une diversification des modes d'entrée. Nous avons aussi observé que virus nu et eBKPyV étaient sensibles à la neutralisation par des anticorps de patient infecté et par des immunoglobulines polyvalentes et que cette neutralisation avait lieu après endocytose. La présence du BKPyV au sein de VE, phénomène portant le nom de "transmission en bloc", pourrait finalement jouer un rôle critique au cours de l'infection primaire, de la persistance, mais aussi de la réactivation du virus et offrir une nouvelle cible dans le développement de nouvelles thérapiesDuring the last years, development of new strong immunosuppressive treatment protocols used in renal transplantation has allowed a better prevention of allograft rejection. However, the induced immunodeficiency leads to the reactivation of multiple viral agents, causative of pathologies with potentially serious consequences such as transplant rejection. The BK virus (BKPyV) is one of these opportunistic viruses. It replicates among one in two transplant patients during the first year post-transplant while more than 80% of the general population is asymptomatic carrier. Unfortunately, its mechanisms of persistence and immune evasion remain misunderstood today. We purified the virus produced in cell culture on a iodixanol gradient and we found that infected cells released two infectious populations of different densities. One of them co-sedimented with extracellular vesicles (EV). This population was less sensitive to protease digestion and its treatment by chloroform leads to the the disappearance of the low-density BKPyV population whereas it increases the high-density population. Electron microscopy allowed us to confirm that EV could transport about ten virions. We also found that the particles included in the EV (eBKPyV), unlike naked particles, are no longer able to agglutinate red blood cells and don’t need sialylated glycan groups to infect cells. The existence of two BKPyV forms could allow diversification of its infection modes. Furthermore, we also observed that naked virus and eBKPyV were both sensitive to neutralization by antibodies from an infected patient and by polyvalent commercial immunoglobulins, with no significant difference, and that this neutralization occured after endocytosis. The presence of BKPyV in extracellular vesicles, a phenomenon called "transmission en bloc", could have a critical incidence during primary infection, persistence but also reactivation of the virus and could be a new target in the development of new therapie

Viruses : vesicle tamers

International audienceThere is a close relationship between viruses and lipid vesicles. The most frequently described concerns enveloped viruses, which acquire their envelope through mechanisms involved in extracellular vesicles (EVs) biogenesis. However, EVs' hijacking is not unique to enveloped viruses. In 2013, a new category of viruses emerged : the quasi-enveloped viruses. These are naked viruses found in vesicles at certain steps of their viral cycle. Actually, several naked viruses, from different families, hijack the production routes of EVs : poliovirus, polyomaviruses, rotavirus, etc. This diversion of EVs confers many advantages : diversification of entry and exit pathways, infectivity improvement and immune evasion. This review will take the reader around this subject

QuantIF: An ImageJ Macro to Automatically Determine the Percentage of Infected Cells after Immunofluorescence

Lynda Handala and Tony Fiore contributed equally to this work.International audienceCounting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI-and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as supplementary data online at MDPI.com and has been developed using ImageJ, a free image processing program that can run on any computer with a Java virtual machine, which is distributed for Windows, Mac, and Linux. It is routinely used in our labs to quantify viral infections in vitro, but can easily be used for other applications that require quantification of labeled cells

Intercellular Transmission of Naked Viruses through Extracellular Vesicles: Focus on Polyomaviruses

Extracellular vesicles have recently emerged as a novel mode of viral transmission exploited by naked viruses to exit host cells through a nonlytic pathway. Extracellular vesicles can allow multiple viral particles to collectively traffic in and out of cells, thus enhancing the viral fitness and diversifying the transmission routes while evading the immune system. This has been shown for several RNA viruses that belong to the Picornaviridae, Hepeviridae, Reoviridae, and Caliciviridae families; however, recent studies also demonstrated that the BK and JC viruses, two DNA viruses that belong to the Polyomaviridae family, use a similar strategy. In this review, we provide an update on recent advances in understanding the mechanisms used by naked viruses to hijack extracellular vesicles, and we discuss the implications for the biology of polyomaviruses

Antibody Responses after a Third Dose of COVID-19 Vaccine in Kidney Transplant Recipients and Patients Treated for Chronic Lymphocytic Leukemia

International audienceThe impact of a third dose of COVID-19 vaccine on antibody responses is unclear in immunocompromised patients. The objective of this retrospective study was to characterize antibody responses induced by a third dose of mRNA COVID-19 vaccine in 160 kidney transplant recipients and 20 patients treated for chronic lymphocytic leukemia (CLL). Prevalence of anti-spike IgG ≥ 7.1 and ≥ 30 BAU/mL after the third dose were 47% (75/160) and 39% (63/160) in kidney transplant recipients, and 57% (29/51) and 50% (10/20) in patients treated for CLL. Longitudinal follow-up identified a moderate increase in SARS-CoV-2 anti-spike IgG levels after a third dose of vaccine in kidney transplant recipients (0.19 vs. 5.28 BAU/mL, p = 0.03) and in patients treated for CLL (0.63 vs. 10.7 BAU/mL, p = 0.0002). This increase in IgG levels had a limited impact on prevalence of anti-spike IgG ≥ 30 BAU/mL in kidney transplant recipients (17%, 2/12 vs. 33%, 4/12, p = 0.64) and in patients treated for CLL (5%, 1/20 vs. 45%, 9/20, p = 0.008). These results highlight the need for vaccination of the general population and the importance of non-medical preventive measures to protect immunocompromised patients

Biology of the BKPyV: An Update

The BK virus (BKPyV) is a member of the Polyomaviridae family first isolated in 1971. BKPyV causes frequent infections during childhood and establishes persistent infections with minimal clinical implications within renal tubular cells and the urothelium. However, reactivation of BKPyV in immunocompromised individuals may cause serious complications. In particular, with the implementation of more potent immunosuppressive drugs in the last decade, BKPyV has become an emerging pathogen in kidney and bone marrow transplant recipients where it often causes associated nephropathy and haemorrhagic cystitis, respectively. Unfortunately, no specific antiviral against BKPyV has been approved yet and the only therapeutic option is a modulation of the immunosuppressive drug regimen to improve immune control though it may increase the risk of rejection. A better understanding of the BKPyV life cycle is thus needed to develop efficient treatment against this virus. In this review, we provide an update on recent advances in understanding the biology of BKPyV

BK polyomavirus in the urine for follow-up of kidney transplant recipients

International audienceOBJECTIVES: After kidney transplantation, human BK polyomavirus (BKPyV) can induce a progressive disease, in three stages: viruria, viremia, and then nephropathy after a few months of viral replication. Therapeutic intervention is recommended when BKPyV is detected in the plasma. The objective of our study was to assess urinary BKPyV nucleic acid test as a predictor for developing viremia. METHODS: We first defined a viruria threshold based on 393 time-matched urine and plasma samples collected after kidney transplantation and then to validate this threshold, we followed-up a cohort of 236 kidney transplant patients. RESULTS: A BKPyV viruria threshold of 6.71 log10 copies/mL best discriminated between plasma-positive and plasma-negative patients (sensitivity: 90.9% (95%CI: 86.5-95); specificity: 90.3% (95%CI: 86.3-94.3); area under the curve: 0.953 (95%CI: 0.933-0.974). In the validation cohort, the risk of developing BKPyV viremia at one year was 16.5% (39/236) and rose to 90.7% (39/43) if BKPyV viruria remained above the threshold of 6.71 for more than one month. CONCLUSION: Sustained BKPyV viruria is a reliable, early marker of patients at high risk of developing BKPyV viremia. This marker should alert the clinician early, and thus allow timely therapeutic intervention

BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission

International audienceMost people are asymptomatic carriers of the BK polyomavirus (BKPyV), but the mechanisms of persistence and immune evasion remain poorly understood. Furthermore, BKPyV is responsible for nephropathies in kidney transplant recipients. Unfortunately, the sole therapeutic option is to modulate immunosuppression, which increases the risk of transplant rejection. Using iodixanol density gradients, we observed that Vero and renal proximal tubular epithelial infected cells release two populations of infectious particles, one of which cosediments with extracellular vesicles (EVs). Electron microscopy confirmed that a single vesicle could traffic tens of viral particles. In contrast to naked virions, the EV-associated particles (eBKPyVs) were not able to agglutinate red blood cells and did not use cell surface sialylated glycans as an attachment factor, demonstrating that different entry pathways were involved for each type of infectious particle. However, we also observed that naked BKPyV and eBKPyV were equally sensitive to neutralization by the serum of a seropositive patient or commercially available polyvalent immunoglobulin preparations, which occurred at a postattachment step, after endocytosis. In conclusion, our work shows a new mechanism that likely plays a critical role during the primary infection and in the persistence, but also the reactivation, of BKPyV. IMPORTANCE Reactivation of BKPyV is responsible for nephropathies in kidney transplant recipients, which frequently lead to graft loss. The mechanisms of persistence and immune evasion used by this virus remain poorly understood, and a therapeutic option for transplant patients is still lacking. Here, we show that BKPyV can be released into EVs, enabling viral particles to infect cells using an alternative entry pathway. This provides a new view of BKPyV pathogenesis. Even though we did not find any decreased sensitivity to neutralizing antibodies when comparing EV-associated particles and naked virions, our study also raises important questions about developing prevention strategies based on the induction or administration of neutralizing antibodies. Deciphering this new release pathway could enable the identification of therapeutic targets to prevent BKPyV nephropathies. It could also lead to a better understanding of the pathophysiology of other polyomaviruses that are associated with human diseases