Routine immunohistochemistry study for polyomavirus BK nephropathy in transplanted kidney biopsies, is it recommended?

BACKGROUND: Early diagnosis and treatment of Polyomavirus BK Nephropathy (PVBKN) is a challenging issue in the management of patients with kidney transplantation. Currently, histopathologic diagnosis is the gold standard method for diagnosis of PVBKN. However, typical viral inclusions may not be found in early stages of the PVBKN and should, instead, be diagnosed using immunohistochemistry (IHC) study. There is no clear consensus about routine IHC tests in the pathologic evaluation of transplanted kidney biopsy samples. MATERIAL AND METHODS: The current study was conducted on transplanted kidney biopsy samples, since 2016 to 2019. The patients who have presented with new onset of allograft dysfunction, at least 2 weeks after transplantation surgery, were included in our study. All these biopsy samples were evaluated with routine renal biopsy stains as well as IHC for SV40 (Simvian Virus 40) antigen. The identification of typical nuclear virus inclusion body and any nuclear positive staining on IHC (≥1+ positive result) were considered as definite evidence of PVBKN. Sensitivity, specificity, Positive Predictive and Negative Predictive Values (PPV and NPV) of histopathologic assessment without IHC study were evaluated. RESULTS: Among 275 included cases, 18 (6.5%) patients with PVBKN were diagnosed. In patients with PVBKN, typical viral inclusions were detected in 14 samples (77.7%), on primary histopathological examination. However, virus-infected cells were identified just after IHC study in 4 (22.2%) of patients. Sensitivity, Specifity, PPV and NPV of morphologic histopathological assay without IHC for detection of PVBKN was 77.7, 100, 100 and 98.4% respectively. CONCLUSION: Routine IHC study for SV40 in all transplanted kidney biopsy samples with new onset of allograft dysfunction, will enhance the diagnostic sensitivity of early stage disease detection.

SV40 seroprevalence in two Latin American countries involved in field trials of candidate oral poliovaccines.

OBJECTIVES: This study sought to determine SV40 seroprevalence in residents of two Latin American countries, Colombia and Nicaragua, which were sites of prelicensure oral poliovaccine (OPV) trials. METHODS: Archival sera were tested for SV40 neutralizing antibody using a virus-specific plaque-reduction assay. Samples included 517 sera from Colombia and 149 sera from Nicaragua. RESULTS: Overall SV40 seroprevalence was 22.8% for Colombian subjects and 12.8% for Nicaraguans. Subgroups of Colombian subjects ranged in frequency of SV40 seropositivity from 10.0% to 38.6%. Birth cohorts both older and younger than the age cohort that contained potential OPV vaccinees from both countries had SV40 antibodies. Gender and ethnicity had no significant effects on SV40 seropositivity. CONCLUSIONS: Inhabitants of both Colombia and Nicaragua had detectable SV40 neutralizing antibody, including those of ages presumably not recipients of potentially SV40-contaminated OPV. This observation provides support for the concept that transmission of SV40 human infections can occur. Frequency of SV40 antibody positivity was elevated over that reported for the US where there was limited use of contaminated OPV. This investigation indicates also that study results of SV40 infections in humans will reflect whether subject populations had probable exposures to contaminated poliovaccines and to environmental conditions favoring cycles of viral transmission.

Unusual cause of fever, vision loss and super refractory status epilepticus in association with simian virus 40 (SV40).

We present a case of a 23-year-old man with history of fever followed by painless complete vision loss, with subsequent new-onset refractory status epilepticus (NORSE). He initially developed bilateral retinitis. A few days later, he started having focal seizures, and subsequently developed super-refractory status epilepticus, requiring anaesthetic agents. MRI brain revealed multifocal cortical and subcortical hyperintensities in occipital and temporoparietal regions without contrast enhancement. MRI repeated a month later showed new lesions with non-visualisation of some previous lesions. Finally, a brain biopsy was done which revealed presence of lymphocytic infiltrate with SV40 inclusions in oligodendrocyte. We propose the affliction of an atypical virus affecting the retina and brain grey and white matter, presenting with NORSE in our patient. Future similar cases and isolation of the virus may help in establishing the conclusive diagnosis.

Human polyomavirus in tonsillar microbiota of an Afghan population group.

Some studies have evidenced the role of human polyomaviruses in head and neck squamous cell carcinoma. BK, JC and SV40 human polyoma viruses are widely recognized as etiological agents associated with malignancies. The aim of this study was to analyse the prevalence of BK, IC and SV40 in tonsillar microbiota in a group of Afghan volunteers. A sample of the tonsillar microbiota was taken from a single site using a sterile oral swab paper stick. A fixed volume of purified DNA from each sample was tested by quantitative real-time polymerase chain reactions to evaluate the number of human cells and the number of viral genomes in each sample. The cell number was evaluated via the quantification of a single copy genomic sequence, which is located in the HMBS locus. The median analyzed cell number in each reaction was 4343 (interquartile range 2074-8470). SV40 was never detected, while prevalence rate was 0.11 (C.I. 0.06-0.20) for BK and 0.10 (C.I. 0.05-0.19) for JC. Further studies are necessary to clarify whether polyomaviruses can be considered a risk factor of oral, oropharyngeal and laryngeal malignancies.

Crystallization, Reentrant Melting, and Resolubilization of Virus Nanoparticles.

Crystallization is a fundamental and ubiquitous process that is well understood in the case of atoms or small molecules, but its outcome is still hard to predict in the case of nanoparticles or macromolecular complexes. Controlling the organization of virus nanoparticles into a variety of 3D supramolecular architectures is often done by multivalent ions and is of great interest for biomedical applications such as drug or gene delivery and biosensing, as well as for bionanomaterials and catalysis. In this paper, we show that slow dialysis, over several hours, of wild-type Simian Virus 40 (wt SV40) nanoparticle solution against salt solutions containing MgCl2, with or without added NaCl, results in wt SV40 nanoparticles arranged in a body cubic center crystal structure with Im3m space group, as a thermodynamic product, in coexistence with soluble wt SV40 nanoparticles. The nanoparticle crystals formed above a critical MgCl2 concentrations. Reentrant melting and resolubilization of the virus nanoparticles took place when the MgCl2 concentrations passed a second threshold. Using synchrotron solution X-ray scattering we determined the structures and the mass fraction of the soluble and crystal phases as a function of MgCl2 and NaCl concentrations. A thermodynamic model, which balances the chemical potentials of the Mg2+ ions in each of the possible states, explains our observations. The model reveals the mechanism of both the crystallization and the reentrant melting and resolubilization and shows that counterion entropy is the main driving force for both processes.

The Landscape of Viral Expression Reveals Clinically Relevant Viruses with Potential Capability of Promoting Malignancy in Lower-Grade Glioma.

Purpose: RNA sequencing (RNA-seq) has recently proved to be effective for revealing novel virus-tumor associations. To get a thorough investigation of virus-glioma associations, we screened viruses in gliomas with RNA-seq data from the Chinese Glioma Genome Atlas project.Experimental Design: In total, 325 samples were enrolled into this study. Reads that failed to map to the human genome were aligned to viral genomes and screened for potential virus-derived transcripts. For quantification, VPKM was calculated according to mapped reads weighted by genome sizes and sequencing depth.Results: We observed that viruses tended to concertedly express in a certain subgroup of patients. Survival analysis revealed that individuals who were infected with Simian virus 40 (SV40) or woolly monkey sarcoma virus (WMSV) had a significantly shorter overall survival than those uninfected. A multivariate Cox proportional hazards model, taking clinical and molecular factors into account, was applied to assess the prognostic value of SV40 and WMSV. Both SV40 and WMSV were independent prognostic factors for predicting patient's survival in lower-grade gliomas. Subsequent gene analysis demonstrated that SV40 was correlated with regulation of transcription, whereas WMSV was correlated with cell-cycle phase, which indicated frequent proliferation of tumor cells.Conclusions: RNA-seq was sufficient to identify virus infection in glioma samples. SV40 and WMSV were identified to be prognostic markers for patients with lower-grade gliomas and showed potential values for targeting therapy. Clin Cancer Res; 23(9); 2177-85. ©2016 AACR.

Fecal Polyomavirus Excretion in Infancy.

Qualitative polymerase chain reaction (PCR) was used to determine the prevalence of fecal excretion of BK virus, JC virus, and simian virus 40 in 1-year-old infants. Overall, 17.8% of 321 specimens from 64.1% of 39 infants were polyomavirus positive. These data suggest that the gastrointestinal tract may be a site of polyomavirus persistence in humans.

Immunologic evidence of a strong association between non-Hodgkin lymphoma and simian virus 40.

BACKGROUND: Non-Hodgkin lymphoma (NHL), the most common cancer of the lymphatic system, is of unknown etiology. The identification of etiologic factors in the onset of NHL is a key event that could facilitate the prevention and cure of this malignancy. Simian virus 40 (SV40) has been considered an oncogenic agent in the onset/progression of NHL. METHODS: In this study, an indirect enzyme-linked immunosorbent assay with 2 synthetic peptides that mimic SV40 antigens of viral capsid proteins 1 to 3 was employed to detect specific antibodies against SV40. Serum samples were taken from 2 distinct cohorts of NHL-affected patients (NHL1 [n = 89] and NHL2 [n = 61]) along with controls represented by oncologic patients affected by breast cancer (BC; n = 78) and undifferentiated nasopharyngeal carcinoma (UNPC; n = 64) and 3 different cohorts of healthy subjects (HSs; HS1 [n = 130], HS2 [n = 83], and HS3 [n = 87]). RESULTS: Immunologic data indicated that in serum samples from NHL patients, antibodies against SV40 mimotopes were detectable with a prevalence of 40% in NHL1 patients and with a prevalence of 43% in NHL2 patients. In HSs of the same median age as NHL patients, the prevalence was 16% for the HS1 group (57 years) and 14% for the HS2 group (65 years). The difference was statistically significant (P < .0001 and P < .001). Interestingly, the difference between NHL1/NHL2 patients and BC patients (40%/43% vs 15%, P < .001) and between NHL1/NHL2 patients and UNPC patients (40%/43% vs 25%, P < .05) was significant. CONCLUSIONS: Our data indicate a strong association between NHL and SV40 and thus a need for innovative therapeutic approaches for this hematologic malignancy.

Neutralizing and IgG antibodies against simian virus 40 in healthy pregnant women in Italy.

OBJECTIVE: Polyomavirus simian virus 40 (SV40) sequences have been detected in various human specimens and SV40 antibodies have been found in human sera from both healthy individuals and cancer patients. This study analyzed serum samples from healthy pregnant women as well as cord blood samples to determine the prevalence of SV40 antibodies in pregnancy. METHODS: Serum samples were collected at the time of delivery from two groups of pregnant women as well as cord bloods from one group. The women were born between 1967 and 1993. Samples were assayed by two different serological methods, one group by neutralization of viral infectivity and the other by indirect ELISA employing specific SV40 mimotopes as antigens. Viral DNA assays by real-time polymerase chain reaction were carried out on blood samples. RESULTS: Neutralization and ELISA tests indicated that the pregnant women were SV40 antibody-positive with overall prevalences of 10.6% (13/123) and 12.7% (14/110), respectively. SV40 neutralizing antibodies were detected in a low number of cord blood samples. Antibody titers were generally low. No viral DNA was detected in either maternal or cord bloods. CONCLUSIONS: SV40-specific serum antibodies were detected in pregnant women at the time of delivery and in cord bloods. There was no evidence of transplacental transmission of SV40. These data indicate that SV40 is circulating at a low prevalence in the northern Italian population long after the use of contaminated vaccines.

Naturally arising strains of polyomaviruses with severely attenuated microRNA expression.

UNLABELLED: Several different polyomaviruses (PyVs) encode microRNAs (miRNAs) that regulate viral as well as host gene expression. However, the functions of polyomaviral miRNAs, particularly during in vivo infection, remain poorly understood. Here we identify rare naturally arising PyVs that are severely attenuated or null for miRNA expression. We identify hypomorphic or null strains for miRNA expression from rhesus macaque simian virus 40 (SV40) and human JC virus. These strains were isolated from immunocompromised hosts and derive from insertions or deletions in the viral DNA that preserve the amino acid reading frame of opposing-strand large T antigen gene. Characterization of the SV40 miRNA hypomorph, K661, shows that it is inhibited at the early miRNA biogenesis step of Drosha-mediated processing. Despite having a nonrearranged enhancer, which a previous study has shown renders some PyVs more susceptible to the autoregulatory activities of the miRNA, restoring miRNA expression to K661 has little effect on virus growth in either immortalized or primary monkey kidney cells. Thus, in addition to any effect of accompanying genomic elements, these results suggest that the cellular context also determines susceptibility to PyV miRNA-mediated effects. Combined, these results demonstrate that polyomaviruses lacking miRNAs can arise infrequently and that the functional importance of polyomaviral miRNAs is context dependent, consistent with an activity connected to the immune status of the host. IMPORTANCE: Diverse virus families encode miRNAs, yet much remains unknown about viral miRNA function and contribution to the infectious cycle. Polyomaviruses (PyVs) are small DNA viruses, long known to be important as etiological agents of rare diseases and valuable models of DNA virus infection. Here, in immunosuppressed hosts, we uncover rare naturally arising variants of different PyVs that have lost the ability to express miRNAs. This represents some of the only known natural viruses to have lost miRNA expression. By probing the biogenesis pathways of these variants, we uncover that miRNA expression is lost via small insertions or deletions that render the transcripts resistant to early steps of miRNA biogenesis while preserving the reading frame of the opposing T antigen transcripts. Overall, our study informs how miRNA genes evolve/devolve in viruses and suggests that miRNA function is exquisitely dependent not only on viral genomic context but also on the cellular and host environment.

Adventitious agents in viral vaccines: lessons learned from 4 case studies.

Since the earliest days of biological product manufacture, there have been a number of instances where laboratory studies provided evidence for the presence of adventitious agents in a marketed product. Lessons learned from such events can be used to strengthen regulatory preparedness for the future. We have therefore selected four instances where an adventitious agent, or a signal suggesting the presence of an agent, was found in a viral vaccine, and have developed a case study for each. The four cases are: a) SV40 in polio vaccines; b) bacteriophage in measles and polio vaccines; c) reverse transcriptase in measles and mumps vaccines; and d) porcine circovirus and porcine circovirus DNA sequences in rotavirus vaccines. The lessons learned from each event are discussed. Based in part on those experiences, certain scientific principles have been identified by WHO that should be considered in regulatory risk evaluation if an adventitious agent is found in a marketed vaccine in the future.

Ethnic differences in polyomavirus simian virus 40 seroprevalence among women in Houston, Texas.

OBJECTIVE: To examine the prevalence and distribution among racial/ethnic groups of polyomavirus SV40 antibodies in women in Houston, Texas. METHODS: Women in three different cohorts reflecting the evolving demographics of Houston were evaluated for frequency of SV40 antibodies using a plaque-reduction neutralization assay. RESULTS: Women in cohort A (enrolled 1972-1973) were 68% (145/212) African-American and 32% Caucasian; the overall frequency of SV40 neutralizing antibodies was 7%. Women in cohort B (enrolled 1975-1977) were Caucasian with an overall frequency of SV40 neutralizing antibodies of 18% (37/211). Women in cohort C (enrolled 1993-1995) were 50% (199/400) African-American, 25% Caucasian, and 25% Hispanic; the overall frequency of SV40 neutralizing antibodies was 10%. Logistic regression analysis for cohort A showed no difference in SV40 neutralizing antibodies with respect to race/ethnicity, pregnancy status, number of previous pregnancies, or history of sexually transmitted diseases. For cohort C, race/ethnicity was identified as a significant factor associated with SV40 neutralizing antibodies, with Hispanics having a seroprevalence of 23% compared to 5-6% in the other two groups (p = 0.01). CONCLUSIONS: A significantly higher SV40 seroprevalence was found among Hispanics than other racial/ethnic groups in the city of Houston. Findings are compatible with a model that certain population groups potentially exposed to SV40-contaminated oral poliovaccines have maintained cycles of SV40 infections.

Mesenchymoproliferative enteropathy associated with dual simian polyomavirus and rhesus cytomegalovirus infection in a simian immunodeficiency virus-infected rhesus macaque (Macaca mulatta).

Opportunistic viral infections are common in simian immunodeficiency virus-infected rhesus macaques and include simian polyomavirus 40 (SV40), which causes interstitial nephritis, pneumonia, meningoencephalitis, and progressive multifocal leukoencephalopathy and rhesus cytomegalovirus (Macacine herpesvirus-3), which is associated with many pathologic manifestations, including the formation of neutrophil-rich gastrointestinal masses. Herein we report the findings of a simian immunodeficiency virus-infected rhesus macaque that presented to necropsy with multiple nodular masses restricted to the proximal jejunum. Histologically, the masses within the lamina propria were composed of abundant, loosely organized, mesenchymal tissue forming broad interlacing whorls and sheets admixed with variable numbers of neutrophils. Cells within the mesenchymoproliferative nodules contained numerous basophilic, intranuclear inclusion bodies with only scattered cytomegalic cells. Immunohistochemistry for rhesus cytomegalovirus and SV40 demonstrated variable numbers of immunopositive cells within the affected nodules. This report is the first description of SV40-associated pathology in the small intestine of a rhesus macaque and highlights the role that opportunistic viral infections can have on gastrointestinal pathology in immunosuppressed rhesus macaques.

Asbestos and SV40 in malignant pleural mesothelioma from a hyperendemic area of north-eastern Italy.

AIMS AND BACKGROUND: Malignant mesothelioma is a fatal cancer of increasing incidence in north-eastern Italy. Together with asbestos, the polyomavirus SV40 was hypothesized to contribute to the onset of malignant mesothelioma. To investigate the putative role of SV40 in the individual susceptibility to asbestos-induced malignant mesothelioma, we conducted a molecular epidemiological study on a series of malignant mesothelioma patients from an area in north-eastern Italy hyperendemic for malignant pleural mesothelioma. METHODS AND STUDY DESIGN: We collected 63 mesothelioma samples from incidence cases of patients diagnosed with malignant pleural mesothelioma in the period 2009-2010. DNA was extracted from patients' tissue biopsies using the BioRobot EZ1 Qiagen workstation. SV40 sequence detection and quantification was performed by specific real time PCR. The 74.6% of the 63 enrolled patients had a history of asbestos exposure. The epithelioid histotype was more prevalent in males (64.0%) and the mixed in females (61.5%) who showed significantly higher cancer co-morbidity (46.1% vs 12%, P = 0.005). SV40 was detected in 22% of MM tumors, with a low viral load. In SV40-positive patients, a threefold increased risk of asbestos exposure was observed, more evident in females (OR 4.32) than in males (OR 1.20). CONCLUSIONS: Our findings indicate that a high prevalence of SV40 was present in malignant mesothelioma incident cases from an area hyperendemic for malignant mesothelioma in north-eastern Italy. Although asbestos is considered the main risk factor in malignant mesothelioma onset, a role for SV40 could be hypothesized.

Assessment of infection with polyomaviruses BKV, JCV and SV40 in different groups of Cuban individuals.

We investigated the frequency of BKV, JCV and SV40 reactivation in three groups of Cuban patients by multiplex nested PCR assay of 40 paraffin-embedded colorectal neoplasm tissues, 113 urine samples, and 125 plasma samples from 27 transplant recipients, and cerebrospinal fluid (CSF) from 67 HIV-1-infected individuals with central nervous system (CNS) disorders. None of these polyomaviruses were detected in colorectal neoplasms. JCV DNA was detected in 2 of 67 patients (2.9%) with CNS disorders, but neither BKV nor SV40 was identified. BKV was found in urine from 38.5% and 28.6% of adult and pediatric transplant recipients, respectively. In adult renal transplant recipients, excretion of BKV in urine was significantly associated with episodes of acute rejection (p=0.012) and with excretion of HCMV in urine (p= 0.008). In Cuba, the polyomaviruses studied here could not be related to colorectal neoplasms, and JCV was rarely detected in CSFs of HIV-1-infected individuals, whilst BKV reactivation was found to occur frequently in organ transplant recipients.

High prevalence of BK polyomavirus sequences in human papillomavirus-16-positive precancerous cervical lesions.

High- and low-grade cervical lesions were analyzed for the presence of polyomavirus (PYV) and human papillomavirus (HPV) sequences. In precancerous cervical lesions, the overall prevalence of PYV sequences was 44% (41/93). Specifically, among the PYV-positive samples, 83% (34/41) tested positive for BK polyomavirus (BKV) sequences, whereas 17% (7/41) were positive for JC-virus. None of the samples were positive for simian virus 40. The presence of BKV DNA in high-grade squamous intraepithelial lesions was confirmed by in situ PCR. BKV sequences were detected more frequently in high-grade squamous intraepithelial lesions, together with the genotype HPV-16. The association of BKV with precancerous cervical lesions suggests that this polyomavirus participates with HPV-16 in the cell transformation process. Alternatively, BKV might multiply better in HPV-16-positive cells from precancerous cervical lesions than in HPV-16-negative cells.

Prospective study of posttransplant polyomavirus infection in renal transplant recipients.

OBJECTIVES: The BK virus is the most common pathogen in renal transplant recipients. Limited information is available regarding JC virus or Simian virus infections in renal transplant recipients. This prospective study sought to investigate the rate of BK virus, JC virus, and Simian virus 40 infections and their influence on allograft function in the early stages after surgery. MATERIALS AND METHODS: In total, 50 renal transplant recipients and 20 healthy blood donors were studied. The BK virus, the JC virus, and the Simian virus 40 were detected by nested qualitative polymerase chain reaction assays in urine and plasma. The difference of glomerular filtration rate among BK virus-infected, JC virus-infected, and uninfected patients was compared using the Kruskal-Wallis test. RESULTS: The polyomavirus viruria was detected in 46% of renal transplant recipients (4% of the BK virus and 42% of the JC virus viruria) and 10% of the healthy blood donors (5% for the BK virus and the JC virus viruria). No polyomavirus viremia was detected. No difference of glomerular filtration rate was found among the 3 groups (chi-square = 0.228; P = .892). CONCLUSIONS: Polyomavirus infections are not uncommon, and the incidence of JC virus infection is much higher in renal transplant recipients than it is in BK virus. Neither BK virus nor JC virus infections appeared to influence graft function in the early stages after surgery.

Rapid titration of viruses by flow cytometry.

Traditionally, the most common methods used to titrate virus stocks are the plaque assay and the hemagglutination assay. The protocol presented here is based on the detection of viral-expressed proteins in infected cells by flow cytometry. It is simpler and more rapid than the traditional plaque-forming assay and it enables high-throughput analyses.