Submit to Viruses Review for Viruses Propose a Special Issue

Journal Browser

► Journal Browser

BK Virus and Transplantation

Print Special Issue Flyer
Special Issue Editors
Special Issue Information
Keywords
Published Papers

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Animal Viruses".

Deadline for manuscript submissions: closed (31 January 2021) | Viewed by 34339

Share This Special Issue

Special Issue Editor

Prof. Dr. Valeria Pietropaolo

E-Mail Website
Guest Editor

Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
Interests: polyomavirus and polyomavirus-associated diseases; polyomavirus reactivation under immunosuppression or during immunomodulatory therapy; polyomavirus involved in cancer transformation; development of in vitro models
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

It is well known that BK virus (BKV)-associated diseases in transplant recipients are an emerging issue and that kidney transplant recipients (KTRs) comprise the patient population that most frequently experiences complications of BKV reactivation. A proportion of these recipients can develop BKV-associated nephropathy (BKVAN), which is associated with a significant risk of allograft loss. In addition, hemorrhagic cystitis (HC) is a well-recognized BKV-associated complication in hematopoietic stem cell transplant (HSCT) recipients.

In this Special Issue of Viruses, we want to explore some aspects related to BKV-associated diseases in transplant recipients, since they are still unclear, as follows:

the mechanism of viral persistence and the conditions that lead to viral reactivation upon immunosuppression;
the role that innate immune mediators play in controlling BKV infection;
a better understanding of viral subtypes and subgroups and their causative roles in the development of clinical syndromes and nephropathy;
the study of new antiviral agents for BKV infection, since the mainstay of managing reactivation is reduction of immunosuppression;
the development of immune-based therapies to combat BKV and BKV-associated complications;
the development of improved diagnostic tools and clinical management strategies, useful for tracing infection trails in epidemiologic investigations.

Prof. Valeria Pietropaolo
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Viruses is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

BK virus
BK virus strains
subtyping
transplantation
immunosuppressive therapy
kidney transplant
BKVAN
haemorrhagic cystitis
noncoding control region (NCCR)
VP1

Published Papers (12 papers)

Download All Papers

Editorial

Jump to: Research, Review, Other

4 pages, 164 KiB

Open AccessEditorial

BK Virus and Transplantation

by Carla Prezioso and Valeria Pietropaolo

Viruses 2021, 13(5), 733; https://0-doi-org.brum.beds.ac.uk/10.3390/v13050733 - 22 Apr 2021

Cited by 3 | Viewed by 1894

Abstract

As guest editors, we are pleased to present this Special Issue on BK virus (BKV) and transplantation with the intention of exploring some aspects related to BKV-associated diseases in transplant recipients, since they are still unclear [...] Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

Research

Jump to: Editorial, Review, Other

11 pages, 754 KiB

Open AccessArticle

High Incidence and Early Onset of Urinary Tract Cancers in Patients with BK Polyomavirus Associated Nephropathy

by Yi-Jung Li, Hsin-Hsu Wu, Cheng-Hsu Chen, Hsu-Han Wang, Yang-Jen Chiang, Hsiang-Hao Hsu, See-Tong Pang, Robert Y. L. Wang and Ya-Chung Tian

Viruses 2021, 13(3), 476; https://0-doi-org.brum.beds.ac.uk/10.3390/v13030476 - 14 Mar 2021

Cited by 5 | Viewed by 1982

Abstract

Over-immunosuppressed kidney transplant recipients are susceptible to malignancies and BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN). This study aimed to verify the association between BKPyV infection and urinary tract cancers (UTC). A total of 244 kidney transplant recipients were enrolled at Chang Gung Memorial Hospital from June 2000 to February 2020. Biopsy-proven BKPyVAN patients (n = 17) had worse kidney function (eGFR: 26 ± 13.7 vs. 47.8 ± 31.0 mL/min/1.73 m²). The 5-year allograft survival rates for patients with and without BKPyVAN were 67% and 93%, respectively (p = 0.0002), while the 10-year patient survival was not different between the two groups. BKPyVAN patients had a significantly higher incidence of UTC compared to the non-BKPyVAN group (29.4% vs. 6.6%). Kaplan–Meier analysis showed that the UTC-free survival rate was significantly lower in BKPyVAN patients, and the onset of UTC was significantly shorter in BKPyVAN patients (53.4 vs. 108.9 months). The multivariate logistic regression analysis demonstrated that age (RR = 1.062) and BKVAN (RR = 6.459) were the most significant risk factors for the development of UTC. Our study demonstrates that BKPyVAN patients have greater allograft losses, higher incidence, a lower cancer-free survival rate, and an earlier onset with a higher relative risk of developing UTC compared to non-BKPyVAN patients. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

14 pages, 2487 KiB

Open AccessArticle

Reactivations of Latent Viral Infections Are Associated with an Increased Thr389 p70S6k Phosphorylation in Peripheral Lymphocytes of Renal Transplant Recipients

by Maxim Cherneha, Johannes Korth, Meike Kaulfuß, Mirko Trilling, Marek Widera, Hana Rohn, Sebastian Dolff, Nina Babel, André Hoerning, Andreas Kribben and Oliver Witzke

Viruses 2021, 13(3), 424; https://0-doi-org.brum.beds.ac.uk/10.3390/v13030424 - 06 Mar 2021

Cited by 2 | Viewed by 1859

Abstract

Reactivations of BK polyoma virus (BKPyV) and human cytomegalovirus (HCMV) frequently cause life- and graft-threatening complications after renal transplantation. Both viruses are dependent on the mTOR pathway for replication. In this study we investigated the association of viral replication with mTOR activity in peripheral lymphocytes of renal transplant recipients. A flow-cytometry based assay for the measurement of Thr389 p70S6k phosphorylation, a surrogate marker of the mTOR pathway was established. Forty-eight adult renal transplant recipients were recruited to measure p70S6k activity in their peripheral blood mononuclear cells. This data set in conjunction with information concerning previous replication of BKPyV and HCMV was examined for correlations. Episodes of BKPyV replication were significantly associated with increased p70S6k phosphorylation in CD4⁺ T lymphocytes (p = 0.0002) and CD19⁺ B lymphocytes (p = 0.0073). HCMV infection of patients with a high-risk HCMV constellation of donor and recipient (D+/R−) was associated with increased p70S6k phosphorylation in CD19⁺ B lymphocytes (p = 0.0325). These associations were found to be independent of the trough levels of the immunosuppressive drugs. Conclusion: P70S6k phosphorylation in peripheral lymphocytes is associated with BKPyV reactivations and to a lesser extent with HCMV infections in renal transplant recipients. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

11 pages, 1186 KiB

Open AccessArticle

BK Polyomavirus Micro-RNAs: Time Course and Clinical Relevance in Kidney Transplant Recipients

by Baptiste Demey, Véronique Descamps, Claire Presne, Francois Helle, Catherine Francois, Gilles Duverlie, Sandrine Castelain and Etienne Brochot

Viruses 2021, 13(2), 351; https://0-doi-org.brum.beds.ac.uk/10.3390/v13020351 - 23 Feb 2021

Cited by 12 | Viewed by 2344

Abstract

Background: Kidney transplant recipients (KTRs) are exposed to a high risk of BK polyomavirus (BKPyV) replication, which in turn may lead to graft loss. Although the microRNAs (miRNAs) bkv-miR-B1-3p and bkv-miR-B1-5p are produced during the viral cycle, their putative value as markers of viral replication has yet to be established. In KTRs, the clinical relevance of the changes over time in BKPyV miRNA levels has not been determined. Methods: In a retrospective study, we analyzed 186 urine samples and 120 plasma samples collected from 67 KTRs during the first year post-transplantation. Using a reproducible, standardized, quantitative RT-PCR assay, we measured the levels of bkv-miR-B1-3p and bkv-miR-B1-5p (relative to the BKPyV DNA load). Results: Detection of the two miRNAs had low diagnostic value for identifying patients with DNAemia or for predicting DNAuria during follow-up. Seven of the 14 KTRs with a sustained BKPyV infection within the first year post-transplantation showed a progressive reduction in the DNA load and then a rapid disappearance of the miRNAs. DNA and miRNA loads were stable in the other seven KTRs. Conclusions: After the DNA-based diagnosis of BKPyV infection in KTRs, bkv-miR-B1-3p and bkv-miR-B1-5p levels in the urine might be valuable markers for viral replication monitoring and thus might help physicians to avoid an excessive reduction in the immunosuppressive regimen. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Graphical abstract

11 pages, 2414 KiB

Open AccessArticle

Reactivation of BK Polyomavirus in Urine Cytology Is Not Associated with Urothelial Cell Carcinoma

by Faisal Klufah, Ghalib Mobaraki, Axel zur Hausen and Iryna V. Samarska

Viruses 2020, 12(12), 1412; https://0-doi-org.brum.beds.ac.uk/10.3390/v12121412 - 08 Dec 2020

Cited by 3 | Viewed by 2876

Abstract

BK polyomavirus (BKPyV) has been associated with some high-grade and special urothelial cell carcinoma (UCC) subtypes in immunosuppressed patients. Here, we evaluated the relationship of BKPyV-positive urine cytology specimens (UCS) with UCC. A large single-institution database was retrospectively searched for UCS positive for decoy cells, suggesting BKPyV infection. These were tested for the presence of BKPyV by PCR and immunohistochemistry (IHC) in urine sediments and formalin-fixed paraffin-embedded (FFPE) tissue samples of UCC. Decoy cells were reported in 30 patients out of the database with 22.867 UCS. Of these 30 patients, 16 (53.3%) had no history of UCC. Six patients out of these 16 had a history of transplantation, 4 had a history of severe chronic medical conditions, and 6 had no chronic disease. The other fourteen patients were diagnosed with either in situ or invasive UCC of the urinary bladder (14/30; 46.6%) prior to the detection of decoy cells in the urine. Nine of these UCC patients received intravesical treatment (BCG or mitomycin) after the first presentation with UCC. However, the clinical data on the treatment of the other five UCC patients was lacking. IHC identified BKPyV-positivity in the urine samples of non-UCC and UCC patients, while no BKPyV positivity was found in FFPE tissues of primary UCCs and metastases. In addition, BKPyV-PCR results revealed the presence of BKPyV DNA in the urine of the UCC cases, yet none in the UCC tissues itself. These data strongly indicate that BKPyV reactivation is not restricted to immunosuppression. It can be found in UCS of the immunocompetent patients and may be related to the intravesical BCG or mitomycin treatment of the UCC patients. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

13 pages, 680 KiB

Open AccessArticle

Viral Genomic Characterization and Replication Pattern of Human Polyomaviruses in Kidney Transplant Recipients

by Lucia Signorini, Maria Dolci, Evaldo Favi, Caterina Colico, Mariano Ferraresso, Rosalia Ticozzi, Giuseppe Basile, Pasquale Ferrante and Serena Delbue

Viruses 2020, 12(11), 1280; https://0-doi-org.brum.beds.ac.uk/10.3390/v12111280 - 09 Nov 2020

Cited by 14 | Viewed by 2041

Abstract

Human Polyomavirus (HPyV) infections are common, ranging from 60% to 100%. In kidney transplant (KTx) recipients, HPyVs have been associated with allograft nephropathy, progressive multifocal leukoencephalopathy, and skin cancer. Whether such complications are caused by viral reactivation or primary infection transmitted by the donor remains debated. This study aimed to investigate the replication pattern and genomic characterization of BK Polyomavirus (BKPyV), JC Polyomavirus (JCPyV), and Merkel Cell Polyomavirus (MCPyV) infections in KTx. Urine samples from 57 KTx donor/recipient pairs were collected immediately before organ retrieval/transplant and periodically up to post-operative day 540. Specimens were tested for the presence of BKPyV, JCPyV, and MCPyV genome by virus-specific Real-Time PCR and molecularly characterized. HPyVs genome was detected in 49.1% of donors and 77.2% of recipients. Sequences analysis revealed the archetypal strain for JCPyV, TU and Dunlop strains for BKPyV, and IIa-2 strain for MCPyV. VP1 genotyping showed a high frequency for JCPyV genotype 1 and BKPyV genotype I. Our experience demonstrates that after KTx, HPyVs genome remains stable over time with no emergence of quasi-species. HPyVs strains isolated in donor/recipient pairs are mostly identical, suggesting that viruses detected in the recipient may be transmitted by the allograft. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

15 pages, 897 KiB

Open AccessArticle

Renal Allograft Biopsies with Polyomavirus BK Nephropathy: Turin Transplant Center, 2015–19

by Elisa Zanotto, Anna Allesina, Antonella Barreca, Francesca Sidoti, Ester Gallo, Paolo Bottino, Marco Iannaccone, Gabriele Bianco, Luigi Biancone, Rossana Cavallo and Cristina Costa

Viruses 2020, 12(9), 1047; https://0-doi-org.brum.beds.ac.uk/10.3390/v12091047 - 20 Sep 2020

Cited by 5 | Viewed by 2577

Abstract

Background: In kidney transplant patients, polyomavirus-associated nephropathy (PVAN) represents a serious complication; the key factor for the development of PVAN is immunosuppression level and modulation of anti-rejection treatment represents the first line of intervention. Allograft biopsy and histology remain the criterion standard for diagnosing PVAN. Methods: All consecutive renal biopsies with the diagnosis of PVAN carried out at the University Hospital City of Health and Science of Turin over a five-years period were studied. Renal allograft biopsy was performed due to renal function alterations associated to medium-high polyomavirus BK (BKV)-DNA levels on plasma specimen. Results: A total of 21 patients underwent a first biopsy to diagnose a possible BKV nephropathy, in 18, a second biopsy was made, in eight, a third biopsy, and finally, three underwent the fourth renal biopsy; following the results of each biopsies, immunosuppressant agents dosages were modified in order to reduce the effect of PVAN. Conclusions: In this study, the clinical and histological features of 21 kidney transplant recipients with BKV reactivation and development of PVAN are described. To date, the only treatment for PVAN consists in the reduction of immunosuppressive agents, constantly monitoring viral load. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

13 pages, 1987 KiB

Open AccessArticle

Indoleamine 2,3-Dioxygenase Is Involved in Interferon Gamma’s Anti-BKPyV Activity in Renal Cells

by Tony Fiore, Elodie Martin, Véronique Descamps, Etienne Brochot, Virginie Morel, Lynda Handala, Fatima Dakroub, Sandrine Castelain, Gilles Duverlie, François Helle and Catherine François

Viruses 2020, 12(8), 865; https://0-doi-org.brum.beds.ac.uk/10.3390/v12080865 - 07 Aug 2020

Cited by 3 | Viewed by 3070

Abstract

Reactivation of BK polyomavirus (BKPyV) infection is frequently increasing in transplant recipients treated with potent immunosuppressants and highlights the importance of immune system components in controlling viral reactivation. However, the immune response to BKPyV in general and the role of antiviral cytokines in infection control in particular are poorly understood. Here, we investigated the efficacy of interferons (IFN) alpha, lambda and gamma with regard to the BKPyV multiplication in Vero cells. Treatment with IFN-gamma inhibited the expression of the viral protein VP1 in a dose-dependent manner and decreased the expression of early and late viral transcripts. Viral inhibition by IFN-gamma was confirmed in human cells (Caki-1 cells and renal proximal tubular epithelial cells). One of the IFN-stimulated genes most strongly induced by IFN-gamma was the coding for the enzyme indoleamine 2,3 dioxygenase (IDO), which is known to limit viral replication and regulates the host immune system. The antiviral activity induced by IFN-gamma could be reversed by the addition of an IDO inhibitor, indicating that IDO has a specific role in anti-BKPyV activity. A better understanding of the action mechanism of these IFN-gamma-induced antiviral proteins might facilitate the development of novel therapeutic strategies. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

22 pages, 3338 KiB

Open AccessArticle

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

by Dorian McIlroy, Mario Hönemann, Ngoc-Khanh Nguyen, Paul Barbier, Cécile Peltier, Audrey Rodallec, Franck Halary, Emilie Przyrowski, Uwe Liebert, Maryvonne Hourmant and Céline Bressollette-Bodin

Viruses 2020, 12(8), 824; https://0-doi-org.brum.beds.ac.uk/10.3390/v12080824 - 29 Jul 2020

Cited by 15 | Viewed by 3472

Abstract

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. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

Review

Jump to: Editorial, Research, Other

21 pages, 1253 KiB

Open AccessReview

BK Polyomavirus Nephropathy in Kidney Transplantation: Balancing Rejection and Infection

by Chia-Lin Shen, Bo-Sheng Wu, Tse-Jen Lien, An-Hang Yang and Chih-Yu Yang

Viruses 2021, 13(3), 487; https://0-doi-org.brum.beds.ac.uk/10.3390/v13030487 - 16 Mar 2021

Cited by 33 | Viewed by 5707

Abstract

BK polyomavirus nephropathy (BKVN) and allograft rejection are two closely-associated diseases on opposite ends of the immune scale in kidney transplant recipients. The principle of balancing the immune system remains the mainstay of therapeutic strategy. While patient outcomes can be improved through screening, risk factors identification, and rapid reduction of immunosuppressants, a lack of standard curative therapy is the primary concern during clinical practice. Additionally, difficulty in pathological differential diagnosis and clinicopathology’s dissociation pose problems for a definite diagnosis. This article discusses the delicate evaluation needed to optimize immunosuppression and reviews recent advances in molecular diagnosis and immunological therapy for BKVN patients. New biomarkers for BKVN diagnosis are under development. For example, measurement of virus-specific T cell level may play a role in steering immunosuppressants. The development of cellular therapy may provide prevention, even a cure, for BKVN, a complex post-transplant complication. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

21 pages, 1602 KiB

Open AccessReview

The Role of HLA and KIR Immunogenetics in BK Virus Infection after Kidney Transplantation

by Marija Burek Kamenaric, Vanja Ivkovic, Ivana Kovacevic Vojtusek and Renata Zunec

Viruses 2020, 12(12), 1417; https://0-doi-org.brum.beds.ac.uk/10.3390/v12121417 - 09 Dec 2020

Cited by 8 | Viewed by 3022

Abstract

BK virus (BKV) is a polyomavirus with high seroprevalence in the general population with an unremarkable clinical presentation in healthy people, but a potential for causing serious complications in immunosuppressed transplanted patients. Reactivation or primary infection in kidney allograft recipients may lead to allograft dysfunction and subsequent loss. Currently, there is no widely accepted specific treatment for BKV infection and reduction of immunosuppressive therapy is the mainstay therapy. Given this and the sequential appearance of viruria-viremia-nephropathy, screening and early detection are of utmost importance. There are numerous risk factors associated with BKV infection including genetic factors, among them human leukocyte antigens (HLA) and killer cell immunoglobulin-like receptors (KIR) alleles have been shown to be the strongest so far. Identification of patients at risk for BKV infection would be useful in prevention or early action to reduce morbidity and progression to frank nephropathy. Assessment of risk involving HLA ligands and KIR genotyping of recipients in the pre-transplant or early post-transplant period might be useful in clinical practice. This review summarizes current knowledge of the association between HLA, KIR and BKV infection and potential future directions of research, which might lead to optimal utilization of these genetic markers. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

Other

Jump to: Editorial, Research, Review

8 pages, 9524 KiB

Open AccessBrief Report

Evidence of BK Polyomavirus Infection in Urothelial but not Renal Tumors from a Single Center Cohort of Kidney Transplant Recipients

by Cinzia Borgogna, Silvia Albertini, Licia Martuscelli, Filippo Poletti, Alessandro Volpe, Guido Merlotti, Vicenzo Cantaluppi, Renzo Boldorini and Marisa Gariglio

Viruses 2021, 13(1), 56; https://0-doi-org.brum.beds.ac.uk/10.3390/v13010056 - 03 Jan 2021

Cited by 5 | Viewed by 2171

Abstract

Emerging evidence indicates that reactivation of BK polyomavirus (BKPyV) in the kidney and urothelial tract of kidney transplant recipients (KTRs) may be associated with cancer in these sites. In this retrospective study of a single center cohort of KTRs (n = 1307), 10 clear cell renal cell carcinomas and 5 urinary bladder carcinomas were analyzed from 15 KTRs for the presence of BKPyV infection through immunohistochemistry and fluorescent in situ hybridization (FISH). Three of these patients had already exhibited biopsy-proven polyomavirus-associated nephropathies (PyVAN). Although the presence of BKPyV large-T antigen was evident in the urothelium from a kidney removed soon after PyVAN diagnosis, it was undetectable in all the formalin-fixed and paraffin-embedded (FFPE) blocks obtained from the 10 kidney tumors. By contrast, large-T antigen (LT) labeling of tumor cells was detected in two out of five bladder carcinomas. Lastly, the proportion of BKPyV DNA-FISH-positive bladder carcinoma nuclei was much lower than that of LT-positive cells. Taken together, our findings further strengthen the association between BKPyV reactivation and cancer development in KTRs, especially bladder carcinoma. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

Journal Menu

Journal Browser

BK Virus and Transplantation

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Published Papers (12 papers)

Editorial

Research

Review

Other

Further Information

Guidelines

MDPI Initiatives

Follow MDPI