-
[show abstract]
[hide abstract]
ABSTRACT: BK polyomavirus (BKV) is classified into four subtypes based on nucleotide variation of a 287 bp typing region in the VP1 protein. Most studies show that subtype I is predominant in different geographic settings, followed by subtype IV. However, BKV subtypes II and III are detected at low rates. In Spain, the prevalence of each subtype is not well known. The aim of this study was to identify the BKV subtypes from a selection of different types of patients and to determine whether different subtypes could be infecting the same patient. A hundred and twenty nine BKV-positive urine samples were selected to amplify and sequence the typing region. Plasma specimens collected at the same time as the urine samples were also studied in 34 patients. A phylogenetic analysis and a study of substitutions in the VP1 protein were carried out with the sequences obtained. Subtype I was the predominant subtype detected in urine (61.2%) and plasma (38.2%) samples followed by subtype II. The analysis of paired samples showed that the subtype found in urine was different from that found in plasma in 10 patients. Fourteen BKV variants based on substitutions in VP1 were identified. The finding of compartmentalized infections involving different subtypes at different sites in some patients might mean specific and different selective pressure in each tissue. The potential involvement in the viral cycle of the different BKV variants found should be analyzed. J. Med. Virol. 85:1402-1408, 2013. © 2013 Wiley Periodicals, Inc.
Journal of Medical Virology 08/2013; 85(8):1402-8. · 2.82 Impact Factor
-
-
[show abstract]
[hide abstract]
ABSTRACT: In vitro studies have demonstrated that bluetongue virus (BTV)-induced vasoactive mediators could contribute to the endothelial cells dysfunction and increased vascular permeability responsible of lesions characteristic of bluetongue (BT) like oedema, haemorrhages and ischaemic necrosis in different tissues. However, few in vivo studies have been carried out to clarify the causes of these lesions. The aim of this study was to elucidate in vivo the pathogenetic mechanisms involved in the appearance of vascular lesions in different organs during BT. For this purpose, tissue samples from goats naturally infected with bluetongue virus serotype 1 (BTV-1) were taken for histopathological and immunohistochemical studies to determine the potential role of proinflammatory cytokines (tumour necrosis factor alpha, TNFα and interleukin one alpha, IL-1α) in the increased vascular permeability and their relationship with the presence of virus. Gross and histopathological examination revealed the presence of vascular damage leading to generalized oedema and haemorrhages. Immunohistochemical studies displayed that endothelial injury may have been due to the direct pathogenic effect of BTV infection on endothelial cells or may be a response to inflammatory mediators released by virus-infected endothelial cells and, possibly, other cell types such as monocytes/macrophages. These preliminary results of what appears to be the first in vivo study of tissue damage in small BT-infected ruminants suggest a direct link between the appearance of vascular changes and the presence of BTV-induced vasoactive cytokines.
Transboundary and Emerging Diseases 05/2012; · 1.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To detect and monitor the sequential changes in virus levels, a reverse transcription quantitative real-time polymerase chain reaction assay using a TaqMan probe was carried out on frozen blood and tissues samples collected from calves experimentally infected with a non-cytopathic Bovine viral diarrhoea virus (BVDV) genotype 1 strain. Blood samples were collected among days 1-14 post-inoculation (p.i). On day 3 p.i, viral RNA was detected in blood samples from six of the eight inoculated animals. Viral RNA was detected in all remaining inoculated animals between 5 and 12 days p.i. The levels of viral RNA increased along the experiment, with a maximal peak between 6 and 9 days p.i. Analysis of virus load in tissues collected from calves euthanized on days 3, 6, 9 and 14 p.i displayed that BVDV was detected on day 3 p.i, being especially abundant in tonsils and ileocaecal valve, highlighting the role of tonsils as the main earliest viral replication sites as well as the principal source for virus spread to other lymphoid tissues and visceral organs. Coinciding with the highest viraemia levels, the highest viral loads were recorded at 9 days p.i. in tonsils, ileal lymph nodes, distal ileum and spleen, showing the main role of these secondary lymphoid organs in the pathogenic mechanisms of BVDV. However, virus levels in the liver and lung increased only towards the end of the infection. This fact could influence in the appearance of bovine respiratory diseases because of the capacity of BVDV for enhancing susceptibility to secondary infections.
Transboundary and Emerging Diseases 12/2011; 59(5):377-84. · 1.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Epidemiological surveys have revealed outbreaks of pandemic influenza A (H1N1) 2009 in several different contexts. Molecular characterization of the influenza virus could help to provide a more accurate description of these outbreaks.
To genotype pandemic influenza A (H1N1) 2009 isolates from an epidemiologically defined nosocomial outbreak.
We sequenced the neuraminidase (NA) and hemagglutinin (HA) influenza A (H1N1) 2009 genes from ten HIV-positive patients involved in an epidemiologically defined outbreak in the Clinical Microbiology and Infectious Diseases (CMID) Department. Sequences were aligned to search for specific genetic features of the involved strain. We also analyzed 37 unrelated influenza A (H1N1) 2009 cases from other hospital departments. All the sequences were used to obtain phylogenetic trees.
Identical genotypic features were shared by nine of the 10 cases initially considered to be involved in the outbreak, but not by the remaining case. These features involved two silent mutations at N385 and V407 in the NA gene and three amino acid substitutions in the HA gene (D225E, A189T, and P300S). Searching for these substitutions in patients with influenza A (H1N1) 2009 hospitalized in other departments during the same period allowed us to identify an additional unsuspected immunocompetent case. The five outbreak-specific substitutions were absent in the remaining 36 unrelated controls. One of the substitutions (P300S) rendered detection of this variant by the CDC protocol inefficient. The other outbreak-specific substitutions (D225E and A189T) were identified at codons that have been analyzed in the context of virulence.
Genotyping is essential to ensure a more accurate description of pandemic influenza A (H1N1) 2009 outbreaks.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 08/2011; 52(2):129-32. · 3.12 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: BK virus (BKV) nephropathy is a common viral infection in renal transplant patients, with a prevalence of 1-9% at approximately 12 months after surgery. While it is widely agreed that reduction of immunosuppression should be the first intervention after diagnosis of BKV infection, there is no consensus on whether calcineurin inhibitors or antiproliferative drugs should be reduced first. Furthermore, target levels of immunosuppressive drugs are poorly defined, as are criteria for replacing one immunosuppressive agent with another. RESULTS: We report our series of 15 renal transplant patients who underwent surgery between September 2004 and March 2010 and who developed BKV infection. The first 8 patients were treated with reduction of immunosuppression; 7 of these patients received cidofovir and 6 received intravenous immunoglobulin. The remaining 7 renal transplant recipients received mammalian target of rapamycin inhibitors (imTOR). In this group, we observed faster and more efficacious BKV clearance in plasma and urine and a steady improvement in allograft function, with no episodes of acute allograft rejection during follow-up. The polymerase chain reaction assay for BKV in urine became positive in 2 patients in whom imTOR were stopped due to severe side effects. CONCLUSIONS: The use of imTOR should be considered a first step in the treatment of renal transplant recipients with BKV infection. In our experience, this change in treatment was safe and resulted in viral clearance.
Transplant Infectious Disease 05/2011; 13(6):584-91. · 2.22 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Background Epidemiological surveys have revealed outbreaks of pandemic influenza A (H1N1) 2009 in several different contexts. Molecular characterization of the influenza virus could help to provide a more accurate description of these outbreaks.Objective To genotype pandemic influenza A (H1N1) 2009 isolates from an epidemiologically defined nosocomial outbreak.Study design We sequenced the neuraminidase (NA) and hemagglutinin (HA) influenza A (H1N1) 2009 genes from ten HIV-positive patients involved in an epidemiologically defined outbreak in the Clinical Microbiology and Infectious Diseases (CMID) Department. Sequences were aligned to search for specific genetic features of the involved strain. We also analyzed 37 unrelated influenza A (H1N1) 2009 cases from other hospital departments. All the sequences were used to obtain phylogenetic trees.Results Identical genotypic features were shared by nine of the 10 cases initially considered to be involved in the outbreak, but not by the remaining case. These features involved two silent mutations at N385 and V407 in the NA gene and three amino acid substitutions in the HA gene (D225E, A189T, and P300S). Searching for these substitutions in patients with influenza A (H1N1) 2009 hospitalized in other departments during the same period allowed us to identify an additional unsuspected immunocompetent case. The five outbreak-specific substitutions were absent in the remaining 36 unrelated controls. One of the substitutions (P300S) rendered detection of this variant by the CDC protocol inefficient. The other outbreak-specific substitutions (D225E and A189T) were identified at codons that have been analyzed in the context of virulence.Conclusions Genotyping is essential to ensure a more accurate description of pandemic influenza A (H1N1) 2009 outbreaks
Journal of Clinical Virology. 01/2011; 52(2).
-
[show abstract]
[hide abstract]
ABSTRACT: The VP7 structural protein is the most abundant of the major core proteins and is highly conserved in all serotypes of bluetongue virus (BTV). The aim of this study was to develop immunohistochemical techniques for the detection of BTV VP7 in Bouin's- and formalin-fixed and paraffin wax-embedded tissues from small ruminants (sheep and goats) naturally infected with BTV. Tissue samples were taken from animals in which BTV infection had been confirmed by reverse transcriptase polymerase chain reaction. Optimal results were obtained by incubation of monoclonal antibody 2E9 on samples fixed with Bouin's solution or neutral buffered formalin. Optimum antigen retrieval for Bouin's-fixed samples was by microwave heating (6 min) of tissue samples in citrate buffer (pH 6.0, 0.01 M), while for formalin-fixed samples a 30 min heating period in pH 9.0 buffer was required. In both species, BTV was mainly detected in the spleen, lymph nodes and lungs; specifically within the arteriolar and capillary endothelial cells, together with macrophages and lymphocytes. The immunohistochemical method described will be a useful tool for future research.
Journal of comparative pathology 02/2010; 143(1):20-8. · 1.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bluetongue virus (BTV), a member of the Orbivirus genus, has contributed to great economical losses in countries across the Mediterranean basin. Although BTV has an African origin, it has been reported in the South of Europe since 1924 when it was first detected in Cyprus. After this first Bluetongue (BT) outbreak, many others followed in most Mediterranean countries, resulting in seven BTV serotypes detected in 17 countries in Central-, South-Europe and North Africa in the last 10 years. Currently, six BTV serotypes (1, 2, 4, 8, 9 and 16) are circulating throughout Central- and Western Europe. The unexpected occurrence of BTV serotype 8 in Central Europe in 2006 and its spread and persistence have evidenced changes in the BTV scenario: new serotypes, never detected in the Mediterranean area so far, are playing a central role in the maintenance of BTV in Europe, and new species of Culicoides are now confirmed to be able to transmit the virus. Therefore, it is necessary to improve and implement specific assays in order to identify the serotypes currently present in different Mediterranean countries in a fast and reliable way. In this study, we present a new gel-based and real-time RT-PCR assay for the detection of BTV serotype 4. The sequence amplified in this test is located within BTV segment 2, a variable region of BTV dsRNA genome encoding the major outer-capsid protein VP2. No cross-reaction has been shown with other genetically or geographically related viruses and the sensitivity of this test allows the detection of 1.5-15 TCID50/ml of BTV.
Transboundary and Emerging Diseases 09/2008; 55(5-6):205-14. · 1.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Al género Orbivirus pertenecen tres virus transmitidos a través de vector, que afectan al ganado domestico y a animales de vida libre (Lengua Azul, Peste Equina Africana y Enfermedad Hemorrágica Epizoótica de los Ciervos), y que han causado en las dos últimas décadas, especialmente el virus de la Lengua Azul (vLA), pérdidas económicas cuantiosas en prácticamente toda Europa y en los países del sur y el este del Mediterráneo. Es importante, por tanto, profundizar en el conocimiento sobre los mecanismos de actuación de estos virus, tomando como base al vLA considerado como el virus modelo para el estudio de este género, y en los mecanismos que modulan la respuesta inmunológica del hospedador para poder mejorar el control de la enfermedad mediante vacunas más eficaces. Hasta el momento se desconocen aspectos tan importantes como la duración de la viremia en animales infectados, el papel de la inmunidad celular desencadenada en el hospedador o los mecanismos de “hibernación” que permitan al vLA “reaparecer” tras un periodo de ausencia del vector. Además, el hecho de que los tres compartan los mismos vectores, hace necesario analizar el riesgo de entrada de los virus de la Peste Equina Africana y dela Enfermedad Hemorrágica Epizoótica de los Ciervos en España, -que actualmente se detectan en los países del África Subsahariana y en el sur y este del Mediterráneo, respectivamente-. Por último, se considera también imprescindible mejorar las técnicas de diagnóstico para el virus de la Enfermedad Hemorrágica Epizoótica de los Ciervos y el virus de la Peste Equina Africana y desarrollar nuevos ensayos basados en las cepas circulantes por el área mediterránea.
Anales de la Real Academia de Ciencias Veterinarias de Andalucía Oriental 21, 51-64 (2008).
-
[show abstract]
[hide abstract]
ABSTRACT: La Lengua Azul, está producida por un virus ARN del género Orbivirus (familia Reoviridae), considerado como el virus prototipo de este género, del que se conocen al menos 24 serotipos diferentes, no todos patógenos, entre los que no existe inmunidad cruzada, lo que difi culta las estrategias de vacunación. En las dos últimas décadas, y más recientemente desde el verano de 2006, esta enfermedad ha provocado importantes pérdidas económicas, no sólo en las zonas de Europa periódicamente afectadas como los países de la cuenca Mediterránea, sino prácticamente en toda Europa. Los planes de vacunación puestos en marcha por las autoridades sanitarias, han revelado la existencia de reacciones adversas, así como la falta de protección de las vacunas en un elevado porcentaje de casos. En este trabajo pretendemos poner de manifi esto la importancia de conocer los mecanismos inmunológicos que se desarrollan tanto en animales infectados como en animales vacunados. Los mecanismos de acción de cada uno de estos serotipos varían completamente dependiendo de la especie y de la raza afectada. Hasta la fecha son escasos los trabajos in vivo que hayan centrado sus esfuerzos en una caracterización pormenorizada de los mecanismos patogénicos y de la repuesta inmune de cada uno de los serotipos patógenos en las principales especies afectadas por la enfermedad. Sólo con el conocimiento de los mecanismos de acción del virus y con el estudio de los mecanismos que controlar y modulan la respuesta inmune podremos desarrollar herramientas (nuevos adyuvantes, aplicación de inmunomoduladores, etc) que nos permitan mejorar la vacunas existentes, reduciendo las reacciones adversas que producen y potenciando su protección.
Anales de la Real Academia de Ciencias Veterinarias de Andalucía Oriental 22, 211-226 (2009).
-
[show abstract]
[hide abstract]
ABSTRACT: The VP7 structural protein is the most abundant of the major core proteins and is highly conserved in all serotypes of bluetongue virus (BTV). The aim of this study was to develop immunohistochemical techniques for the detection of BTV VP7 in Bouin's- and formalin-fixed and paraffin wax-embedded tissues from small ruminants (sheep and goats) naturally infected with BTV. Tissue samples were taken from animals in which BTV infection had been confirmed by reverse transcriptase polymerase chain reaction. Optimal results were obtained by incubation of monoclonal antibody 2E9 on samples fixed with Bouin's solution or neutral buffered formalin. Optimum antigen retrieval for Bouin's-fixed samples was by microwave heating (6 min) of tissue samples in citrate buffer (pH 6.0, 0.01 M), while for formalin-fixed samples a 30 min heating period in pH 9.0 buffer was required. In both species, BTV was mainly detected in the spleen, lymph nodes and lungs; specifically within the arteriolar and capillary endothelial cells, together with macrophages and lymphocytes. The immunohistochemical method described will be a useful tool for future research.
Journal of Comparative Pathology.
