Polyomaviruses BK and JC DNA infection in peripheral blood cells from blood donors

ABSTRACT Objectives: To investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. Methods: The study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at −70 °C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. Results: Of 250 blood samples, 66 (26.4%) were positive for BKV DNA (146-34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65-21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. Discussion: Our study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30-39 years compared to other age ranges.

BK virus salivary shedding and viremia in renal transplant recipients

Abstract Objectives: This study aimed to verify the presence of polyomavirus BK (BKPyV) in the saliva of kidney transplant recipients and to correlate it with blood viremia. Material and Methods: We have conducted a cross-sectional study with a sample involving 126 renal transplant recipients. 126 samples of saliva and 52 samples of blood were collected from these patients. Detection and quantification of BKPyV were performed using a real-time PCR. To compare the presence of BKPyV in blood and saliva, the binomial proportion test was used. To verify associations between salivary shedding BKPyV and post-transplant periods (in months), the Mann-Whitney test was used. Spearman's correlation was used to correlate the viral load in the saliva with blood of kidney transplant recipients. Results: The mean age of the study group was 51.11±12.45 years old, and 69 participants (54.8%) were female, with a mean post-transplantation time of 4.80±6.04 months. BKPyV was quantified in several samples of saliva and blood, with medians of 1,108 cp/mL and 1,255 cp/mL, respectively. Only 16/52 (30.8%) participants presented BKPyV in blood, and 59/126 (46.8%) excreted the virus in saliva (p=0.004). BKPyV shedding was found in patients at a shorter post-transplantation period (3.86±5.25, p=0.100). A weak correlation was observed between viral quantification in saliva and blood (Spearman's correlation coefficient=0.193). Conclusion: The results of this study suggested that, although saliva excretes more BKPyV than blood, there is no reliable correlation between salivary shedding and blood viremia, showing two independent compartments of viral replication.

Quantitative detection of BK virus in kidney transplant recipients: a prospective validation study / Detecção quantitativa de vírus BK em receptores de transplante renal: um estudo prospectivo de validação

Abstract Introduction: BK virus (BKV) infection in renal transplant patients may cause kidney allograft dysfunction and graft loss. Accurate determination of BKV viral load is critical to prevent BKV-associated nephropathy (BKVAN) but the cut-off that best predicts BKVAN remains controversial. Objective: To evaluate the performance of a commercial and an in-house qPCR test for quantitative detection of BK virus in kidney transplant recipients. Methods: This was a prospective study with kidney transplant recipients from two large university hospitals in Brazil. Patients were screened for BKV infection every 3 months in the first year post-transplant with a commercial and an in-house real time polymerase chain reaction (qPCR) test. BKVAN was confirmed based on histopathology. The area under the curve for plasma qPCR was determined from receiver operating characteristic analysis. Results: A total of 200 patients were enrolled. Fifty-eight percent were male, 19.5% had diabetes mellitus, and 82% had the kidney transplanted from a deceased donor. BKV viremia was detected in 32.5% and BKVAN was diagnosed in 8 patients (4%). BKVAN was associated with viremia of 4.1 log copies/mL, using a commercial kit. The cut-off for the in-house assay was 6.1 log copies/mL. The linearity between the commercial kit and the in-house assay was R2=0.83. Conclusion: Our study shows that marked variability occurs in BKV viral load when different qPCR methodologies are used. The in-house qPCR assay proved clinically useful, a cheaper option in comparison to commercial qPCR kits. There is an urgent need to make BKV standards available to the international community.

Resumo Introdução: A infecção pelo vírus BK (BKV) em pacientes de transplante renal pode levar a disfunção do aloenxerto renal e perda do enxerto. A determinação precisa da carga viral do BKV é fundamental para prevenir a nefropatia associada ao BKV (BKVAN), mas o ponto de corte de melhor valor preditivo para BKVAN ainda é foco de debates. Objetivo: Avaliar o desempenho de um teste de qPCR comercial e outro desenvolvido internamente para detecção quantitativa de vírus BK em receptores de transplante renal. Métodos: O presente estudo prospectivo incluiu receptores de transplante renal de dois grandes hospitais universitários no Brasil. Os pacientes foram testados para infecção por BKV a cada três meses no primeiro ano pós-transplante com um teste comercial de reação em cadeia de polimerase quantitativa em tempo real (qPCR) e outro desenvolvido internamente. A presença de BKVAN foi confirmada com base na histopatologia. A área sob a curva para o qPCR plasmático foi determinada a partir da análise da característica de operação do receptor. Resultados: Um total de 200 pacientes foram incluídos. Cinquenta e oito por cento eram do sexo masculino, 19,5% tinham diabetes mellitus e 82% tiveram seus rins transplantados de doadores falecidos. Viremia de BKV foi detectada em 32,5% dos pacientes e oito (4%) foram diagnosticados com BKVAN. BKVAN foi associada a viremia de 4,1 log cópias/mL usando o kit comercial. O corte para o ensaio interno foi de 6,1 log cópias/mL. A linearidade entre o kit comercial e o ensaio interno foi R2 = 0,83. Conclusão: Nosso estudo demonstrou uma acentuada variabilidade na carga viral de BKV quando diferentes metodologias de qPCR foram utilizadas. O ensaio interno de qPCR mostrou-se clinicamente útil, além de ser uma opção menos onerosa em relação aos kits comerciais de qPCR. Há uma necessidade urgente de se definir padrões de BKV para a comunidade internacional.

Human polyomaviruses and cancer: an overview

The name of the family Polyomaviridae, derives from the early observation that cells infected with murine polyomavirus induced multiple (poly) tumors (omas) in immunocompromised mice. Subsequent studies showed that many members of this family exhibit the capacity of mediating cell transformation and tumorigenesis in different experimental models. The transformation process mediated by these viruses is driven by viral pleiotropic regulatory proteins called T (tumor) antigens. Similar to other viral oncoproteins T antigens target cellular regulatory factors to favor cell proliferation, immune evasion and downregulation of apoptosis. The first two human polyomaviruses were isolated over 45 years ago. However, recent advances in the DNA sequencing technologies led to the rapid identification of additional twelve new polyomaviruses in different human samples. Many of these viruses establish chronic infections and have been associated with conditions in immunosuppressed individuals, particularly in organ transplant recipients. This has been associated to viral reactivation due to the immunosuppressant therapy applied to these patients. Four polyomaviruses namely, Merkel cell polyomavirus (MCPyV), Trichodysplasia spinulosa polyomavirus (TSPyV), John Cunningham Polyomavirus (JCPyV) and BK polyomavirus (BKPyV) have been associated with the development of specific malignant tumors. However, present evidence only supports the role of MCPyV as a carcinogen to humans. In the present review we present a summarized discussion on the current knowledge concerning the role of MCPyV, TSPyV, JCPyV and BKPyV in human cancers.

Evaluation of the predisposition and clinical impact of BK virus replication in kidney transplant patients

ABSTRACT The BK virus (BKV) produces a subclinical kidney infection in immunocompetent individuals. However, viremia may occur in kidney transplant patients with ongoing immunosuppression. BKV-associated nephropathy (BKVN) has no specific treatment and is a leading cause of organ transplant loss. In this study, we evaluated the predisposition and the clinical impact of BKV replication in kidney transplant patients during post-transplant monitoring in a reference institution in Brazil. Demographic, clinical and laboratory data generated during routine outpatient follow-up were retrospectively collected. BK viremia was investigated using real-time polymerase chain reaction. Of the 553 participants, 7.4% (n = 41) presented BKV replication. Of these, 16 (39%) lost their kidney graft and interstitial nephritis was identified on kidney biopsy in 50% of the cases. Among the evaluated variables, only the use of the immunosuppressant mycophenolate sodium was identified as a risk factor for viremia (OR 7.96; 95% CI 2.35 to 26.98). The graft survival estimate in BKV-positive patients was significantly reduced (24.8% vs. 85.6%) after 10 years of transplantation. We concluded that defining predisposing factors remains an important challenge for the prevention and control of BKV activity following kidney transplantation, especially considering the development of BKVN and its strong effect on graft maintenance.

Bovine Herpesvirus 4 in Parana State, Brazil: case report, viral isolation, and molecular identification

Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus. This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cow’s uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after HindIII digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes HindIII and BamHI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of BamHI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group.

Infecção oral pelo HPV em mulheres com lesão escamosa de colo uterino no sistema prisional da cidade de São Paulo, Brasil / Oral infection by the Human Papilloma Virus in women with cervical lesions at a prison in São Paulo, Brazil

O carcinoma de cabeça e pescoço é 6ª maior causa de mortes por neoplasia no mundo. Nas últimas décadas, tem-se associado a relação da infecção pelo Papilomavírus Humano (HPV) e seu envolvimento na etiologia desta doença, bem como acontece com o câncer de colo de útero. OBJETIVO: A caracterização molecular dos tipos de HPV diagnosticados na mucosa oral de mulheres que apresentavam alterações citológicas compatíveis com lesão escamosa no colo uterino. MÉTODOS: Foram estudadas 409 amostras cérvico-vaginais e de cavidade oral de mulheres internas no Presídio Feminino da cidade de São Paulo. A correlação entres lesões cervicais e orais foram avaliadas em 27 mulheres que apresentavam lesões pré-malignas e malignas no colo uterino pela caracterização molecular dos tipos de HPV por PCR/ RFLP e Sequenciamento. RESULTADOS: Das 27 (6,67%) amostras compatíveis com LSIL e HSIL no colo uterino, 22 (81,48%) apresentaram infecção pelo HPV de alto risco oncogênico, sendo o HPV 59 o mais prevalente, dentre elas, três amostras (11,1%) evidenciaram alterações celulares compatíveis com displasia leve na cavidade oral. CONCLUSÃO: Nosso estudo sugere uma relação entre o desenvolvimento de lesões da cavidade oral e a infecção pelo HPV, independentemente do tipo viral presente.

Carcinoma of the head and neck is the 6th cause of death by cancer in the world. In recent decades the human papillomavirus (HPV) has been implicated in the etiology of this disease. OBJECTIVE: To characterize the types of HPV detected in the oral mucosa in women with cytological abnormalities suggesting intraepithelial squamous lesions in the uterine cervix. METHODS: four-hundred-nine cervical-vaginal and oral pap-smears of women interned in a Female Prison in São Paulo were examined. The relationship between cervical and oral lesion was analyzed by PCR/RFLP and DNA sequencing. RESULTS: Of 27 (6.67%) specimens showing cervical cytological abnormalities suggesting LSIL and HSIL, 22 (81.48%) had oncogenic high-risk HPV infection, of which HPV 59 was the most prevalent. Three (11.1%) samples showed cytological changes suggesting mild dysplasia in the oral cavity. CONCLUSION: Our study suggests an association between carcinoma of the oral cavity and HPV infection, regardless of the virus type.

Prevalence of human papillomavirus and Epstein-Barr virus DNA in penile cancer cases from Brazil

Penile cancer is a potentially mutilating disease. Although its occurrence is relatively rare worldwide, penile cancer rates can be high in developing countries. A few studies have been conducted on the involvement of human papillomavirus (HPV) in penile carcinoma, which have found HPV present in 30-70 percent of penile malignant lesions, with a higher prevalence of HPV 16 and 18. It has been assumed that cofactors, such as Epstein-Barr virus (EBV) infections, may play a role in the progression of penile neoplasia. The aim of this study was to determine HPV and EBV prevalence in 135 penile malignant lesions from Brazilian men through the use of MY09/11 polymerase chain reaction (PCR), type-specific PCR and restriction fragment length polymorphism analysis. HPV prevalence among the men tested was 60.7 percent. Of the men who tested positive, 27 presented with HPV 16 (29.7 percent), five with HPV 18 (5.5 percent), 21 with HPV 45 (23.1 percent) and nine with HPV 6 (9.9 percent). Seven mixed infections were detected (9.2 percent), while 11 cases remained untyped (13.4 percent). Regarding EBV positivity, 46.7 percent of the samples contained EBV DNA with EBV-1 as the most prevalent type (74.6 percent). More than 23 percent of the men were co-infected with both HPV and EBV, while 35 percent presented exclusively with HPV DNA and 20 percent presented only with EBV DNA. Penile carcinoma aetiology has not been fully elucidated and the role of HPV and EBV infections individually or synergistically is still controversial. Hence, more studies are needed to determine their possible role in carcinogenesis.

Cáncer cérvicouterino y virus del papiloma humano / Cervical cancer and human papillomavirus

El cáncer cérvicouterino (CaCu) es la segunda causa de muerte por cáncer en mujeres de todo el mundo, a pesar de la implementación de la citología de cérvix para su prevención. Esto se debe a la baja sensibilidad y especificidad de la prueba, lo cual apoya a un cambio urgente en la forma de tamizaje para su detección. Ahora se sabe que la infección persistente por virus del papiloma humano de alto riesgo (HR-HPV) es la causa de la totalidad de los casos de CaCu. En la actualidad se están utilizando vacunas frente a dos (Bivalente: HPV-16 y HPV-18) o cuatro (Tetravalente: HPV-6 HPV-11, HPV-16 y HPV-18) de las cepas de HR-HPV que causan la mayoría de los casos de CaCu. El propósito de este artículo es proporcionar una revisión de las características principales del virus y de los mecanismos que se echan a andar bajo la infección persistente de las células cervicales, lo cual conduce a la proliferación desordenada y a la malignización de las células infectadas. Es necesario que el virus se integre al genoma de la célula epitelial para que inicie la expresión de las oncoproteínas virales E6 y E7 lo cual conducirá al desarrollo del CaCu.

Cervical cancer (CC) is the second cause of death for cancer in women worldwide in spite of the implementation of cervix cytology screenings for its prevention. The low sensibility and specificity of the test reduce the potential benefits of these screenings and supports urgent improvements in early detection tests for CC. It is now known that persistent infection with the high-risk human papiloma virus (HR-HPV) is the causal agent of almost all cases of CC. HR-HPV vaccines effective against two (Bivalent: HPV-16 and HPV-18) or four (Tetravalent: HPV-6 HPV-11, HPV-16 and HPV-18) strains that are responsible of the majority of the CC cases have been licensed in several countries. The present study aims to provide a review of the principal characteristics of the HR-HPV virus and of the mechanisms that take to the persistent infection of the cervical cells leading to abnormal proliferation and malignancy. It is necessary that the virus integrates into the genome of the epithelial cell to initiates the expression of the E6 and E7 viral oncoproteins which will lead to the development of the CC.

Polyoma BK virus: an emerging opportunistic infectious agent of the human central nervous system

BK virus, a double-stranded DNA virus, is a member of the Polyomaviridae family which is known to infect humans. Clinical evidence of disease is mostly encountered in immunosuppressed individuals such as AIDS patients or those who undergo renal or bone marrow transplantation where complications associated with BKV infection manifest commonly as a polyomavirus nephropathy or hemorrhagic cystitis, respectively. Recent evidence suggests that in addition to the JC virus (the other member of the same family known to be strongly neurotropic and responsible for the progressive multifocal leukoencephalopathy), BK virus can infect and cause clinically relevant disease in the human central nervous system. In this mini-review, an analysis of the literature is made. A special focus is given to alert clinicians to the possibility of this association during the differential diagnosis of infections of the central nervous system in the immunocompromised host.

Infecção pelo papilomavírus humano: etiopatogenia, biologia molecular e manifestações clínicas / Human papillomavirus infection: etiopathogenesis, molecular biology and clinical manifestations

O papilomavírus humano (HPV) é um vírus DNA que apresenta tropismo por células epiteliais, causando infecções na pele e nas mucosas. A replicação do HPV ocorre no núcleo das células escamosas e o seu ciclo de vida é diretamente relacionado ao programa de diferenciação da célula hospedeira. Até o momento, foram completamente caracterizados cerca de 100 tipos diferentes de HPVs e há um grande número adicional de tipos ainda não sequenciados. Além de ser o responsável por lesões benignas de pele e mucosas, o HPV também está envolvido no desenvolvimento de diversos tumores cutaneomucosos: doença de Bowen, cânceres de pele não melanoma e carcinomas genitais. Esta revisão aborda as características do HPV, quadros cutâneos e mucosos benignos e malignos causados por ele e os principais métodos empregados em sua detecção e tipagem.

Human papillomavirus (HPV) is a DNA virus that presents tropism for epithelial cells, causing infections of the skin and mucous membranes. Replication of HPV occurs in the nuclei of squamous cells and its life cycle is directly related to the differentiation program of the host cell. To date, nearly 100 different types of HPV have been characterized and there is a large number of other types that have not been sequenced yet. Besides being responsible for benign lesions of the skin and mucous membranes, HPV is also involved in the development of various mucocutaneous tumors: Bowen's disease, non-melanoma skin cancers and genital carcinomas. This review discusses the characteristics of HPV, malignant and benign mucous and skin manifestations caused by HPV, besides the main methods of detection and typing of the virus.

Papilomavírus humano e neoplasia cervical: [revisão] / Human papillomavirus and cervical neoplasia: [revision]

Human papillomavirus (HPV) has been established as an important etiological factor for the development of cervical cancer. This DNA virus primarily infects the epithelium and can induce benign and malignant lesions of the mucous membranes and skin. Some HPVs are considered high risk due to their role in malignant progression of cervical tumors. Genital HPV infections are common and usually transient among young sexually active women. Only a small fraction of infected women develop cervical cancer, implying the involvement of environmental and genetic cofactors in cervical carcinogenesis. Classification, virology, pathology, natural history, epidemiological features of genital HPV infection, and future prospects for cervical cancer prevention with HPV vaccines will be reviewed here.

O papilomavírus humano (HPV) é um fator etiológico bem estabelecido para o câncer cervical. Esse vírus de DNA infecta primariamente o epitélio e pode induzir lesões benignas ou malignas na pele e na mucosa. Alguns HPVs são considerados de alto risco, responsáveis pela progressão das lesões precursoras até câncer cervical. A infecção genital pelo HPV é comum em mulheres jovens e geralmente é transitória. Uma pequena proporção de mulheres infectadas desenvolve câncer cervical, implicando o envolvimento de fatores ambientais e fatores genéticos na carcinogênese. Essa revisão aborda a estrutura viral, classificação e patologia do HPV, história natural e fatores de risco para neoplasia cervical e perspectivas futuras com a vacina anti-HPV.

Diagnóstico y terapia del virus papiloma humano / Diagnosis and treatment of human papilloma virus

Tanto el diagnóstico como el tratamiento de la infección producida por el virus papiloma humano y el cáncer asociado a este virus, nos plantean uno de los mayores desafíos en la última década. Las principales dificultades radican en la identificación del genotipo viral, la ausencia de una terapia antiviral efectiva y las altas tasas de recurrencia y persistencia a pesar de la terapia empleada. Se presenta un resumen de la terapia disponible en la actualidad.

The identification and treatment of human papillomavirus (HPV) infections and HPV-associated neoplasm are complex. Difficulties in diagnosis and treatment of HPV-associated diseases arise from inabilities to detect HPV efficiently, the lack of specific antiviral drugs active against HPV and the high rates of recurrence and persistence of HPV infections after treatment. We present a review of therapies for HPV infections.

Human papillomavirus genotypes in women with cervical cytological abnormalities from an area with high incidence of cervical cancer

La relación entre la infección por los virus papiloma humanos (HPVs) de alto riesgo y el cáncer de cuello de útero ha sido bien demostrada. En el Nordeste de Argentina se observa una alta incidencia de esta patología y en consecuencia se estima una alta prevalencia de infección por HPV. A fin de identificar los genotipos de HPV presentes en el área, asociados a casos de lesiones malignas y premalignas de cuello de útero, se estudiaron 53 muestras ecto-endo cervicales de mujeres con alteraciones citohistológicas residentes permanentes de las ciudades de Resistencia y Corrientes. De las 53 pacientes estudiadas, 34 resultaron positivas para HPV (64.2 por ciento), correspondiendo la mayor frecuencia a HPV-16 (32.3 por ciento), seguido por HPV-58 (14.7 por ciento), HPV-6, -18 y -45 (5.9 por ciento), -33, -52, -53, -54, -56, -66, -MM4 y -LVX100 (2.9 por ciento). Además, se encontraron 5 casos de infecciones mixtas causadas por mas de un genotipo, lo que resulta de importancia ya que representan el 14.7 por ciento del total de los casos positivos. Los resultados demuestran que, además de HPV-16 y -18 que son los genotipos de alto riesgo de mayor prevalencia a nivel mundial, otros como el HPV-45 y -58 y los casos de infecciones múltiples son frecuentes en mujeres del nordeste argentino, lo que podría constituir un rasgo epidemiológico de importancia regional y ser de utilidad en el futuro en los programas de vacunación.

La dicotomia de los virus polioma: infeccion litica o induccion de neoplasias? / The paradox of polyomaviruses: lytic infection or tumor induction?

Los virus Polioma murinos provocan infecciones líticas en cultivos de células de ratón y transforman in vitro células de rata a través de la interacción de su oncogén mT con diversos reguladores celulares. Luego de su inoculación en ratones neonatos inducen neoplasias epiteliales y mesenquimáticas. Se ha propuestoque las cepas de polioma más oncogénicas son aquellas que previamente replican más en el ratón. Sin embargo, a nivel de una sola célula la infección lítica y la transformación deberían ser mutuamente excluyentes.En cada neoplasia han sido descriptos 3 tipos celulares según expresen el DNA viral solo o concomitantementecon la proteína mayor de la cápside VP1, o que no contengan DNA viral ni VP-1. En nuestro laboratorio detectamos la existencia de un cuarto tipo celular en las neoplasias, en el que se expresa la totalidad del genoma viral pero no ocurre el ensamblaje, probablemente por alteraciones en la fosforilación de VP-1. Se discuten los mecanismos de migración intracelular de Polioma, la diseminación en el ratón y los factores que podrían estarinvolucrados en la inducción de neoplasias o en la infección lítica inducidas por el virus.

Human papillomavirus prevalence, viral load and cervical intraepithelial neoplasia in HIV-infected women

HIV-infected women from S o Paulo city were enrolled in a cross-sectional study on Human Papillomavirus (HPV) and cervical intraepithelial neoplasia (CIN) prevalence and their association with laboratory markers of AIDS, namely HIV viral load and CD(4)(+) cell counts. A cervical specimen was collected and submitted to Hybrid Capture, a test for HPV viral load determination. HPV-DNA was detected in 173 of 265 women (64.5). Twenty (7.5) women were infected by one or more low-risk viruses, 89 (33) by one or more high-risk viruses, and 64 (24) harbored at least one HPV type from each risk group. Abnormal smears were observed in 19 of the patients, though there were no invasive carcinomas. Severely immunosuppressed patients (CD(4)/microL <100) were at the greatest risk of having a cytological abnormality and a high high-risk HPV viral load.