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.

BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission.

Most 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.

[BK-virus and pathophysiology of associated diseases]. / Le virus BK et physiopathologie des maladies associées.

BK virus (BKV) is a widely distributed polyomavirus in the world population. It is the causative agent of BKV-associated nephropathy in kidney transplant recipients and hemorrhagic cystitis in bone marrow transplant patients. To date, there is no specific antiviral treatment against BKV. A better understanding of the pathophysiology of BKV-associated diseases, especially in immunocompromised patients, may contribute to the development of novel preventive and therapeutic strategies. After a detailed description of the genomic characteristics of the virus, its replication cycle and available model systems, the pathophysiological and immune mechanisms involved in BKV infection are developed and discussed in this review.

BK virus: Current understanding of pathogenicity and clinical disease in transplantation.

BK polyomavirus (BKV) is an important cause of graft loss in renal transplant recipients that continues to pose a significant challenge to clinicians due to its frequently unpredictable onset, persistence, and the lack of effective antiviral agents or prevention strategies. This review covers our current understanding of epidemiology, viral transmission and disease progression, and treatment and prevention strategies that have been used to manage this disease.

Metagenomic Virome Sequencing in Living Donor and Recipient Kidney Transplant Pairs Revealed JC Polyomavirus Transmission.

BACKGROUND: Before kidney transplantation, donors and recipients are routinely screened for viral pathogens using specific tests. Little is known about unrecognized viruses of the urinary tract that potentially result in transmission. Using an open metagenomic approach, we aimed to comprehensively assess virus transmission in living-donor kidney transplantation. METHODS: Living kidney donors and their corresponding recipients were enrolled at the time of transplantation. Follow-up study visits for recipients were scheduled 4-6 weeks and 1 year thereafter. At each visit, plasma and urine samples were collected and transplant recipients were evaluated for signs of infection or other transplant-related complications. For metagenomic analysis, samples were enriched for viruses, amplified by anchored random polymerase chain reaction (PCR), and sequenced using high-throughput metagenomic sequencing. Viruses detected by sequencing were confirmed using real-time PCR. RESULTS: We analyzed a total of 30 living kidney donor and recipient pairs, with a follow-up of at least 1 year. In addition to viruses commonly detected during routine post-transplant virus monitoring, metagenomic sequencing detected JC polyomavirus (JCPyV) in the urine of 7 donors and their corresponding recipients. Phylogenetic analysis confirmed infection with the donor strain in 6 cases, suggesting transmission from the transplant donor to the recipient, despite recipient seropositivity for JCPyV at the time of transplantation. CONCLUSIONS: Metagenomic sequencing identified frequent transmission of JCPyV from kidney transplant donors to recipients. Considering the high incidence rate, future studies within larger cohorts are needed to define the relevance of JCPyV infection and the donor's virome for transplant outcomes.

Impact of donor BK polyomavirus replication on recipient infections in living donor transplantation.

BACKGROUND: Multiple risk factors for BK polyomavirus (BKPyV) replication after kidney transplantation have been described. Here, we investigated the impact of living donors' urinary BKPyV shedding and recipients' BKPyV antibody status pre-transplant on BKPyV replication during the first year post-transplantation. METHODS: We assessed a cohort of living kidney donors and their paired recipients (n = 121). All donors were tested before transplantation, and recipients were tested before and after transplantation for BKPyV viruria and viremia. BKPyV-specific serology was assessed in all recipients at transplantation. RESULTS: Ten of 121 donors (8.3%) had urinary BKPyV shedding pre-transplant, none had viremia. Overall, 33 (27.3%) recipients developed viruria after transplantation: 7 had received a kidney from a donor with BK viruria (7/10 positive donors) and 26 had received a kidney from a donor without BK viruria (26/111 negative donors; P = .0015). Fifteen (12.4%) recipients developed BK viremia after transplantation: 3 received a kidney from a donor with viruria (3/10 positive donors, 30%) and 12 received a kidney from a donor without viruria (12/111 negative donors, 11%; P = .08). One patient developed proven nephropathy. Ninety-one percent of recipients were seropositive for BKPyV. No relationship between recipients' sero-reactivity at transplantation and post-transplant BKPyV replication was observed. Pre-transplant donor urinary shedding was an independent risk factor for post-transplant BKPyV replication. CONCLUSION: Screening living kidney donors for BKPyV can identify recipients at higher risk for BKPyV replication after transplantation who may benefit from intensified post-transplant screening and treatment strategies.

A Naturally Transmitted Epitheliotropic Polyomavirus Pathogenic in Immunodeficient Rats: Characterization, Transmission, and Preliminary Epidemiologic Studies.

We report the identification, pathogenesis, and transmission of a novel polyomavirus in severe combined immunodeficient F344 rats with null Prkdc and interleukin 2 receptor gamma genes. Infected rats experienced weight loss, decreased fecundity, and mortality. Large basophilic intranuclear inclusions were observed in epithelium of the respiratory tract, salivary and lacrimal glands, uterus, and prostate gland. Unbiased viral metagenomic sequencing of lesioned tissues identified a novel polyomavirus, provisionally named Rattus norvegicus polyomavirus 2 (RatPyV2), which clustered with Washington University (WU) polyomavirus in the Wuki clade of the Betapolyomavirus genus. In situ hybridization analyses and quantitative polymerase chain reaction (PCR) results demonstrated viral nucleic acids in epithelium of respiratory, glandular, and reproductive tissues. Polyomaviral disease was reproduced in Foxn1rnu nude rats cohoused with infected rats or experimentally inoculated with virus. After development of RatPyV2-specific diagnostic assays, a survey of immune-competent rats from North American research institutions revealed detection of RatPyV2 in 7 of 1,000 fecal samples by PCR and anti-RatPyV2 antibodies in 480 of 1,500 serum samples. These findings suggest widespread infection in laboratory rat populations, which may have profound implications for established models of respiratory injury. Additionally, RatPyV2 infection studies may provide an important system to investigate the pathogenesis of WU polyomavirus diseases of man.

Evaluation of the Gastrointestinal Tract as Potential Route of Primary Polyomavirus Infection in Mice.

BACKGROUND: Detection of Polyomavirus (PyV) DNA in metropolitan rivers worldwide has led to the suggestion that primary viral infection can occur by the oral route. The aim of this study was to test this notion experimentally. METHODS: Mouse PyV (MPyV) was used to infect C57BL/6J mice by the nasal or intragastric route. Viral load kinetics was studied 3, 7, 10, 14, 21 and 28 days post-infection (dpi) using quantitative PCR. RESULTS: Following nasal infection, MPyV DNA was readily detected in many organs including lung, heart, aorta, colon, and stool with viral loads in the range of 10(3)-10(6) copies/mg wet weight that peaked 7-10 dpi. Complete viral clearance occurred in the serum and kidney by 28 dpi, while clearance in other organs was partial with a 10-100 fold decrease in viral load. In contrast, following intragastric infection peak detection of PyV was delayed to 21 dpi, and viral loads were up to 3 logs lower. There was no detectable virus in the heart, colon, or stool. CONCLUSIONS: The intragastric route of MPyV infection is successful, not as efficacious as the respiratory route, and associated with delayed viral dissemination as well as a lower peak MPyV load in individual organs.

Polyomavirus and Naturally Occuring Neuroglial Tumors in Raccoons (Procyon Lotor).

Polyomavirus (PyV) infections are widespread in human populations and, although generally associated with silent persistence, rarely cause severe disease. Among diseases convincingly associated with natural PyV infections of humans, there are remarkably different tissue tropisms and outcomes, including progressive multifocal leukoencephalopathy, transient or progressive nephropathy, and cancer. The variable character and unpredictable outcomes of infection attest to large gaps in our basic understanding of PyV biology. In particular, the rich history of research demonstrating the oncogenic potential of PyVs in laboratory animals begs the question of why cancer is not more often associated with infection. Raccoon polyomavirus (RacPyV), discovered in 2010, is consistently identified in neuroglial tumors in free-ranging raccoons in the western United States. Exposure to RacPyV is widespread, and RacPyV is detected in tissues of raccoons without tumors. Studying the relationship of RacPyV with its natural host is a unique opportunity to uncover cogent cellular targets and protein interactions between the virus and its host. Our hypothesis is that RacPyV, as an intact episome, alters cellular pathways within neural progenitor cells and drives oncogenesis.

Viral Origin, Clinical Course, and Renal Outcomes in Patients With BK Virus Infection After Living-Donor Renal Transplantation.

BACKGROUND: BK virus (BKV) nephropathy remains the main cause of renal graft loss after living-donor renal transplantation. The aim of the study was to investigate the source and factors influencing the course of BKV infection. METHODS: We investigated 214 living donor-recipient pairs. Urine and blood of donors and recipients were tested by qPCR for the presence of BKV DNA before and after transplantation; genotyping of BKV subtypes was performed. RESULTS: Eighty-five recipients (40%) had posttransplant BK viruria including 61 with additional viremia and 22 with nephropathy. Pretransplant urinary BKV shedding of donor or recipient was a significant risk factor for posttransplant viruria and viremia (OR, 4.52; CI, 2.33-8.77; P < 0.0001) and nephropathy (OR, 3.03; CI, 1.16-7.9; P = 0.02). In the BKV nephropathy group, urine and blood became BKV positive earlier than in the group with viruria and viremia. Renal function was worse in BKV-nephropathy compared with BKV-negative patients beginning at transplantation. Comparing BKV subtypes of donor and recipient before with the subtype of the infected recipient after transplantation, donor-derived transmission was identified in 24 of 28 corresponding pairs. BKV subtype IV had a higher prevalence in recipients with BKV nephropathy than in those with viruria and viremia (P = 0.045). CONCLUSIONS: Pretransplant urinary BKV shedding of donor and recipient is a risk for posttransplant infection. Donor-derived BKV transmission is an important mode of infection. BKV subtype IV may be one of the viral determinants. Early BKV positivity of urine and blood indicates later BKV nephropathy. Decreased renal function may favor BKV infection.

Human leukocyte antigen Cw7-mediated protection against polyoma BK virus in renal transplant recipients who received grafts from antigen-positive donors.

OBJECTIVES: Nephropathy from BK virus is an increasing problem in renal transplant recipients and has been correlated with newer immunosuppressive agents and the decline in acute rejection rates. We aimed to evaluate the effect of BK virus-positive kidney donors on the outcome of kidney transplant recipients after mean follow-up 21 months. MATERIALS AND METHODS: Among 18 kidney donors with BK virus in blood and urine, 5 donors were fit for donation. Clinical information was reviewed for the 5 kidney transplant recipients who received kidney allografts from these donors (mean donor age, 35 ± 3 y). RESULTS: All recipients except 1 were women (mean age, 49.4 ± 4.2 y; body weight, 68.2 ± 4 kg, followup, 21.6 ± 4 mo). All patients except 1 received antithymocyte globulin induction, and all 5 patients received steroids, tacrolimus, and mycophenolate mofetil as maintenance therapy. Ureter stenting was a routine procedure in each case. Human leukocyte antigen Cw7 was detected in 4 of 5 recipients, and the fifth case, the antigen was detected in the donor. At last follow-up, all patients were enjoying functioning grafts without recurrence of BK virus infection. CONCLUSIONS: Polyoma BK virus-positive people can be accepted safely for kidney donation, especially with a possible protective role of human leukocyte antigen Cw7.

The polyomavirus puzzle: is host immune response beneficial in controlling BK virus after adult hematopoietic cell transplantion?

BK virus (BKV), a ubiquitous human polyomavirus, usually does not cause disease in healthy individuals. BKV reactivation and disease can occur in immunosuppressed individuals, such as those who have undergone renal transplantation or hematopoietic cell transplantation (HCT). Clinical manifestations of BKV disease include graft dysfunction and failure in renal transplant recipients; HCT recipients frequently experience hematuria, cystitis, hemorrhagic cystitis (HC), and renal dysfunction. Studies of HCT patients have identified several risk factors for the development of BKV disease including myeloablative conditioning, acute graft-versus-host disease, and undergoing an umbilical cord blood (uCB) HCT. Although these risk factors indicate that alterations in the immune system are necessary for BKV pathogenesis in HCT patients, few studies have examined the interactions between host immune responses and viral reactivation in BKV disease. Specifically, having BKV immunoglobulin-G before HCT does not protect against BKV infection and disease after HCT. A limited number of studies have demonstrated BKV-specific cytotoxic T cells in healthy adults as well as in post-HCT patients who had experienced HC. New areas of research are required for a better understanding of this emerging infectious disease post HCT, including prospective studies examining BK viruria, viremia, and their relationship with clinical disease, a detailed analysis of urothelial histopathology, and laboratory evaluation of systemic and local cellular and humoral immune responses to BKV in patients receiving HCT from different sources, including uCB and haploidentical donors.

Donor origin of BKV replication after kidney transplantation.

BACKGROUND: BK virus associated nephropathy (BKVN) leads to renal allograft dysfunction and loss. However, it is still unclear whether BKV replication in the transplant recipient is a result of reactivation in the recipient's native kidneys or whether BKV originates from the donor kidney. STUDY DESIGN: Urine of 249 donor/recipient pairs was investigated for the presence of BKV-DNA by qPCR before living transplantation (Tx) and consecutively after Tx. In BKV positive samples, the VP1 typing region (TR) and, in case of the presence of sufficient amount of DNA, the complete VP1 gene, the NCCR and a fragment of the Large T-antigen were sequenced and compared between donors and corresponding recipients before and after Tx. RESULTS: In 20 pairs, sequencing of the BKV TR succeeded in donors and corresponding recipients after Tx. The derived sequences were completely identical in donor and post-Tx recipient samples. For comparison, identical TR sequences were detected in only 24% of 1068 randomly assembled pairs. This difference was statistically highly significant (p<0.0001, Fisher's exact test). Furthermore, all VP1, Large T-antigen and NCCR BKV sequences were also identical between donors and corresponding post-Tx recipients. In two of the 20 donor/recipient pairs, VP1 TR sequencing was also successful from the recipient before Tx. In both cases the sequence differed from the sequence detected in donor and recipient after Tx giving further evidence that recipient BKV was replaced by donor BKV after Tx. CONCLUSIONS: Our study for the first time provides evidence of BKV donor origin in renal transplant recipients.

Oncogenic Papillomavirus and Polyomavirus in Water Environments: Is There a Potential for Waterborne Transmission?

Waterborne exposure to human viruses through contact with sewage-contaminated water environments can result in infections associated with a wide range of illnesses. Gastrointestinal symptoms are the most commonly encountered manifestations of waterborne viral illness. Respiratory diseases, neurological diseases and paralysis can also occur. Whether viral infections resulting in health outcomes like cancer might also be transmitted by the waterborne route is unknown. Recently, viruses belonging to two oncogenic groups-Human Papillomaviruses (HPVs) and Human Polyomaviruses (HPyVs)-have been detected in urban sewages worldwide. The latter have also been identified in other water environments. HPVs are epitheliotropic viruses responsible for several diseases of skin and mucosae, from common warts to squamous intraepithelial lesions that can either heal or progress to invasive carcinoma of the cervix, vulva, vagina, penis, anus or oropharynx. Human PyVs infect different tissues and organs, causing infections that are usually subclinical in immunocompetent individuals but can be serious in immunocompromised hosts. These pathogens belong to a family of DNA tumour viruses. Merkel cell polyomavirus, a HPyV identified in recent years, has attracted much attention due to its link with a rare and aggressive form of human cancer. Merkel cell carcinoma, the incidence of which has tripled over the past two decades. JC polyomavirus and BK polyomavirus are also potentially oncogenic. The observed abundance and wide dissemination of HPVs and HPyVs in water environments strongly suggest the need to shed light on the fate of these viruses in water environments and to elucidate their potential for waterborne transmission. Such information is essential for the improvement of wastewater management programs in terms of both sewage treatment and water quality surveillance.

Merkel cell polyomavirus infection occurs during early childhood and is transmitted between siblings.

Merkel cell polyomavirus (MCPyV) is thought to be the etiological agent of Merkel cell carcinoma, but little is known about its distribution and modes of transmission. We conducted seroepidemiological surveys in more than 1000 individuals, from two populations from Cameroon. Overall MCPyV seroprevalence was high in both populations (>75% in adults). Data from the first population, comprising mainly children, indicated that MCPyV infections mostly occurred during early childhood, after the disappearance of specific maternal antibodies. Results from the second family-based population provided evidence for familial aggregation of MCPyV infection status. We observed significant sib-sib correlation (odds ratio=3.42 [95% CI 1.27-9.19], p=0.014), particularly for siblings close together in age, and a trend for mother-child correlation (OR=2.71 [0.86-8.44], p=0.08). Overall, our results suggest that MCPyV infection is acquired through close contact, possibly involving saliva and/or the skin, especially between young siblings and between mothers and their children.

Serological cross-reactivity between human polyomaviruses.

Until 2006, BKPyV and JCPyV were the only known human polyomaviruses. A third polyomavirus, simian virus 40 whose natural host is the macaque was accidently introduced into man because of contaminated poliovirus vaccines, although there is epidemiological evidence that SV40 may be transmitted between man independently from contaminated vaccines. Since 2007, 10 new human polyomaviruses have been identified: KIPyV, WUPyV, Merkel cell polyomavirus, trichodysplasia spinulosa-associated polyomavirus, and human polyomaviruses 6, 7, 9, 10, STL, and 12. Moreover, the DNA of the monkey lymphotropic polyomavirus has been amplified from human peripheral blood. Seroepidemiological studies frequently based on the presence of antibodies against the major capsid protein VP1 or virus-like particles indicate that most human adults have been exposed to many, if not all, human polyomaviruses. However, because of the high amino acid sequence identity between VP1 of some human polyomaviruses, cross-reactivity of antibodies is occasionally observed. In addition, human sera possess reactivity against VP1 of polyomaviruses from other species, suggesting serological cross-reaction with known or closely related, yet unidentified human polyomaviruses and/or the possibility of zoonotic transmission. Thus, current serological results should be interpreted with caution, and controls excluding cross-reactivity with other polyomaviruses are required.

Monitoring of KI and WU polyomaviruses in hematopoietic stem cell transplant patients.

Primary infection with KIPyV and WUPyV polyomaviruses occurs early in childhood followed by lifelong persistence in the body. Polyomavirus reactivation can occur in the presence of impaired immunity as in hematological malignancies or during immunosuppresssion induced by medications. In this study, reactivation of KIPyV and WUPyV was monitored by conventional PCR in plasma samples of 26 stem cell transplant patients and in 26 related bone marrow donors. Plasma samples from transplant patients were collected immediately after the end of conditioning regimen and up to 270 days after transplant. All plasma samples from transplant patients were negative for KIPyV and WUPyV DNA. Instead, KIPyV DNA was detected in two bone marrow donors. There was no evidence of KIPyV transmission from the donor to the recipient. The data suggest that detection of KIPyV in plasma is sporadic and that KPIyV and WUPyV do not affect the post-transplant clinical course. However, further studies on a larger sample size and more sensitive PCR methods are needed to confirm these observations.

The trichodysplasia spinulosa-associated polyomavirus: virological background and clinical implications.

Trichodysplasia spinulosa-associated polyomavirus (TSPyV) is a new species of the family Polyomaviridae that was discovered in 2010. TSPyV infects humans and is associated with the development of a rare disease called trichodysplasia spinulosa. Trichodysplasia spinulosa is a skin disease of severely immunocompromised hosts characterized by follicular distention and keratotic spine formation especially on the face. Electron microscopy, immunohistochemistry, and viral load measurements indicate an etiological role of active TSPyV infection in the development of this disease. This review will address virological and pathogenic properties of TSPyV, as well as epidemiologic, diagnostic, and therapeutic aspects of TSPyV infection.