RESUMEN

Quantitative real-time PCR (qPCR) is increasingly being used for the detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for the validation and standardization of such tests is currently lacking. Therefore, the present study was initiated by three Office International des Epizooties (OIE) reference laboratories and three collaborating laboratories to measure the interlaboratory variability of six already developed and available BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to six testing centers. Based on qualitative results, the overall agreement among all six laboratories was moderate. However, significant variability in the measurement of the BLV proviral DNA copy number was observed among different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy numbers without harmonization. Further standardization of different factors (i.e., utilization of unified protocols and unique calibrators) should increase interlaboratory agreement.

Asunto(s)

Leucosis Bovina Enzoótica/diagnóstico , Virus de la Leucemia Bovina/fisiología , Provirus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Carga Viral/métodos , Animales , Bovinos , Pruebas Diagnósticas de Rutina/normas , Laboratorios/normas , Virus de la Leucemia Bovina/genética , ARN Viral/genética , Carga Viral/normas

RESUMEN

Orthopoxvirus infections appear to be rare in South American Camelids, because only a few cases have been reported in the literature. Based on a generalized infection with cowpox virus in an alpaca, the clinical symptoms, laboratory diagnostic findings and the pathological changes are described. The case history showed a long treatment because of chronic skin lesions. The main clinical symptom was miliary papules over the entire skin. Furthermore, a bilateral mucopurulent conjunctivitis occurred as well as excessive salivation due to a severe erosive-ulcerative stomatitis. Although the animal received intensive treatment, it died 8 days after admission to the clinic. During necropsy, an erosive-ulcerative laryngitis as well as a necrotising pneumonia and lymphadenitis were observed. Histopathological examination of representative organ samples led to the diagnosis of a suspected orthopoxvirus infection. Electron microscopy and quantitative polymerase chain reaction (qPCR) of tissue samples confirmed this diagnosis. The virus could be isolated in tissue culture and a PCR with subsequent nucleotide sequencing identified cowpox virus as the causative agent for this generalised infection.

Asunto(s)

Camélidos del Nuevo Mundo/virología , Virus de la Viruela Vacuna/aislamiento & purificación , Viruela Vacuna/veterinaria , Animales , Viruela Vacuna/virología

RESUMEN

In early 2006, the highly pathogenic avian influenza virus (HPAIV) H5N1 of the Asian lineage caused the death of wild aquatic birds in Northern Germany. In the mainly affected areas, a trans-species transmission of HPAIV H5N1 to mammals occurred between birds and domestic cats and 1 Stone Marten (Martes foina), respectively. Here, we report lesions and distribution of influenza virus antigen in 3 cats infected naturally with HPAIV H5N1 A/swan/Germany/R65/06. The hemagglutinin partial nucleotide sequences of the viruses were genetically closely related to a H5N1 HPAIV obtained from a dead Whooper Swan (Cygnus cygnus) of the same area. At necropsy, within the patchy dark-red and consolidated lungs, there was granulomatous pneumonia caused by Aelurostrongylus sp. Histologically, the main findings associated with influenza in all cats were bronchointerstitial pneumonia and marked random hepatic necrosis. In addition, all animals displayed lymphoid necrosis in the spleen and Peyer's patches and necrosis of the adrenal cortex. Immunohistochemically, nucleoprotein of HPAIV was present intralesionally in the lungs, liver, adrenal glands, and lymphoid tissues. Oropharyngeal swabs were shown to be suited to detect HPAIV by quantitative real-time polymerase chain reaction (RT-PCR) in these cats, despite the paucity of influenza virus antigen in the upper respiratory tract by means of immunohistochemistry. The results show that outdoor cats in areas affected by HPAIV in wild birds are at risk for lethal infection. In conclusion, hepatic necrosis was, besides bronchointerstitial pneumonia, the primary lesion, suggesting that in naturally infected cats, damage to the liver plays an important role in the pathogenesis of H5N1 influenza.

Asunto(s)

Aves/virología , Enfermedades de los Gatos/patología , Enfermedades de los Gatos/virología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/veterinaria , Glándulas Suprarrenales/patología , Animales , Animales Salvajes/virología , Enfermedades de los Gatos/inmunología , Enfermedades de los Gatos/transmisión , Gatos , Femenino , Tracto Gastrointestinal/patología , Inmunohistoquímica , Hígado/patología , Pulmón/patología , Masculino , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología

RESUMEN

To develop an animal model resembling natural asymptomatic Borna disease virus (BDV) infections, BDV He/80 rat brain homogenate was passaged four times in adult SJL/J mice. Within 12 months of observation, mice did not develop overt signs of disease. Nucleotide sequencing of the rat isolate and the mouse isolates at the fourth passage revealed no difference in the deduced amino acids. Viral RNA was found in brain, heart, kidney, lung, liver, and urinary bladder. Infectious virus was isolated from brain, but also from heart and lung tissue. Immunohistochemically, BDV was demonstrated in nerves in the abdominal cavity, ganglion coeliacum, and adrenal glands, but not in organ parenchyma. Occasionally, viral RNA was detected in mononuclear blood cells.

Asunto(s)

Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/crecimiento & desarrollo , Encéfalo/virología , Replicación Viral , Animales , Anticuerpos Antivirales/sangre , Enfermedad de Borna/inmunología , Enfermedad de Borna/patología , Encéfalo/patología , Sistema Nervioso Entérico/patología , Sistema Nervioso Entérico/virología , Ratones , Ratones Endogámicos

RESUMEN

Retinae of Borna disease virus (BDV)-infected Lewis rats were investigated with emphasis on long-term changes in organotypic tissue organization and glia-neuron relationship. Virus inoculation was attained via intracerebral BDV injection. Following survival times ranging between two and eight months, the retinal thickness was reduced up to one third of that of controls. Photoreceptor segments were completely extinguished and the number of neurons was dramatically reduced. The typical laminar organization of the retina was largely dissolved. Electron microscopy revealed severe spongy degeneration. Large numbers of activated microglia and macrophages were found, both cell types performing very active phagocytosis. The microglial cells expressed an extraordinary phenotype as characterized by large numbers of processes, with some of them penetrating the endfeet of Müller cells and others establishing highly complex interdigitations with vacuolized swellings and endings of neuronal processes. Müller cells were not reduced in number but displayed clear indications of gliosis such as alterations in the immunoreactivity for filament proteins and glutamine synthetase, significantly thickened stem processes, and an altered pattern of K(+) currents in patch-clamp recordings. These findings demonstrate for the first time long-term neuron-glia interactions in the retina of BDV-infected rats. Moreover, the data contribute to our knowledge on structural and functional alterations accompanying persisting virus infection in the central nervous system.

Asunto(s)

Enfermedad de Borna/patología , Virus de la Enfermedad de Borna/patogenicidad , Retina/patología , Enfermedades de la Retina/patología , Animales , Antígenos Virales/metabolismo , Enfermedad de Borna/fisiopatología , Virus de la Enfermedad de Borna/metabolismo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Potenciales de la Membrana/fisiología , Microglía/patología , Microglía/ultraestructura , Microglía/virología , Microscopía Electrónica , Técnicas de Cultivo de Órganos , Células Fotorreceptoras/patología , Células Fotorreceptoras/ultraestructura , Células Fotorreceptoras/virología , Ratas , Ratas Endogámicas Lew , Retina/ultraestructura , Retina/virología , Enfermedades de la Retina/fisiopatología , Enfermedades de la Retina/virología , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/ultraestructura , Células Ganglionares de la Retina/virología

RESUMEN

Cells of the peripheral blood of experimentally and naturally borna disease virus (BDV)-infected animals and of human psychiatric patients and healthy individuals were analyzed for the presence of viral RNA using a BDV-p40-specific nested reverse transcription-polymerase chain reaction (RT-PCR). The assay proved to be highly sensitive as 10 RNA molecules were reproducibly amplified. BDV RNA was detected in blood cells of experimentally infected immunocompetent mice and rats. Mice were persistently infected without showing clinical signs of borna disease (BD), whereas the rats suffered from acute BD. Among 19 horses examined, five were positive for viral RNA in the blood. In a flock of sheep with a history of BD, 1 out of 25 clinically healthy animals was positive. BDV RNA was also detected in cells of the peripheral blood of 10 out of 27 selected humans with psychiatric disorders, and in 2 out of 13 healthy individuals. Remarkably, BDV-specific RNA was present in some cases in the absence of BDV-specific antibodies. Sequence analysis of PCR products confirmed the specificity of the amplification system. The presence of BDV RNA in the blood of naturally and experimentally BDV-infected individuals may point to an incidental but relevant role of blood for the spread of BDV in the infected organism, as well as for the transmission of BDV to other individuals.

Asunto(s)

Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/aislamiento & purificación , Leucocitos Mononucleares/virología , ARN Viral/sangre , Animales , Secuencia de Bases , Enfermedad de Borna/genética , Humanos , Inyecciones Intraventriculares , Leucocitos , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Sensibilidad y Especificidad

RESUMEN

Neuron-glia interactions in the Borna disease virus (BDV)-infected rat retina were investigated with emphasis on the ultrastructural characterization of degenerative alterations in the ganglion cell and photoreceptor layer. Immuno- and cytochemical techniques were applied to label microglia, macrophages and Müller (macroglial) cells. Four weeks after intracerebral infection of adult rats, the total thickness of the retina was considerably diminished, primarily due to the loss of photoreceptor segments and ganglion cells. A gradual reduction of both plexiform layers was also observed. There was a remarkable increase in the number of microglial cells, predominantly in the ganglion cell and the inner plexiform layers. Ultrastructural analysis confirmed that microglia, but also macrophages, were involved in phagocytosis accompanying severe neuronal degeneration in the ganglion cell and the photoreceptor layer. In contrast, Müller cells showed moderate morphological and cytochemical alterations, indicating that Müller cells play only a minor role in early stages of BDV-induced retinitis. Monitoring neuron-glia interactions in BDV-induced retinopathy, combined with the application of different protocols of immunosuppression effecting the BDV virus and/or the microglia, might help to establish specific strategies to suppress BDV-induced neuronal degeneration.

Asunto(s)

Enfermedad de Borna/patología , Virus de la Enfermedad de Borna/aislamiento & purificación , Neuroglía/patología , Neuronas/patología , Retina/patología , Retinitis/patología , Animales , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/inmunología , Infecciones Virales del Ojo/patología , Infecciones Virales del Ojo/virología , Inmunohistoquímica , Macrófagos/inmunología , Macrófagos/patología , Microglía/patología , Microglía/ultraestructura , Microscopía Electrónica , Degeneración Nerviosa , Neuroglía/fisiología , Neuroglía/ultraestructura , Neuronas/fisiología , Neuronas/ultraestructura , Ratas , Ratas Endogámicas Lew , Retina/ultraestructura , Retina/virología , Células Ganglionares de la Retina/patología , Retinitis/virología , Coloración y Etiquetado/métodos

Asunto(s)

Virus de la Inmunodeficiencia Felina , Precursores de Proteínas/genética , Proteínas del Envoltorio Viral/genética , Animales , Gatos , Mutagénesis Sitio-Dirigida , Eliminación de Secuencia , Relación Estructura-Actividad

RESUMEN

The cellular tropism of the feline immunodeficiency virus (FIV) is affected by changes in variable region 3 (V3) of the surface (SU) envelope glycoprotein (Verschoor, E. J., et al., J. Virol. 69:4752-4757, 1995). By using high-dose DNA transfection, an FIV molecular clone with a non-CRFK-tropic V3 acquired the ability to replicate in CRFK cells. A single point mutation from a methionine to a threonine in the ectodomain of its transmembrane (TM) envelope glycoprotein was responsible for this change in viral tropism. This substitution is located in the putative SU interactive region, between the fusion peptide and the membrane-spanning region. Our results show that this region of the TM envelope glycoprotein constitutes an additional determinant for cell tropism.

Asunto(s)

Glicoproteínas/fisiología , Virus de la Inmunodeficiencia Felina/fisiología , Proteínas del Envoltorio Viral/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Gatos , ADN , Glicoproteínas/genética , Metionina/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Envoltorio Viral/genética

Asunto(s)

Interleucina-12/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Gatos , Clonación Molecular , Datos de Secuencia Molecular , Alineación de Secuencia

RESUMEN

A competitive reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify RNA of feline immunodeficiency virus (FIV) in cats. The assay uses in vitro synthesized RNA as a competitive internal control. The synthesized RNA has a 22-base deletion with respect to the wild-type sequence. PCR products were quantitated by densitometric analysis of a digitized image of the ethidium bromide stained gel. Viral RNA concentrations in the plasma of two cats experimentally infected with FIV strain UT113 were followed for 32 weeks; peak copy numbers (2.3 x 10(4) and 1.3 x 10(4) per ml, respectively) were reached 11 weeks after subcutaneous injection of ten 50% cat infectious doses. With rising antibody titers against FIV-gag and FIV-env gene products, the amount of FIV RNA in plasma decreased. Nine asymptomatic cats that had been experimentally infected 3.5 to 4.5 years earlier had copy numbers between 5.6 x 10(3) and 4.3 x 10(4) per ml. Cats treated for six weeks with 9-(2-phosphonylmethoxy-propyl)-2,6-diaminopurine [PMPDAP] (20 mg/kg body weight s.c. three times a week) showed a significant decrease of RNA copy numbers in plasma. This quantitative competitive RT-PCR will be useful to study the pathogenesis of the FIV infection, to evaluate the effectiveness of vaccines and to monitor antiviral and immunomodulating drugs.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida del Felino/virología , Virus de la Inmunodeficiencia Felina/aislamiento & purificación , ARN Viral/sangre , Viremia/virología , Animales , Anticuerpos Antivirales/sangre , Relación CD4-CD8 , Gatos , Virus de la Inmunodeficiencia Felina/genética , Reacción en Cadena de la Polimerasa/veterinaria

RESUMEN

A competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed to quantify RNA of feline immunodeficiency virus (FIV) in cats. The assay uses in vitro synthesized RNA derived from the gag region of the FIV genome as a competitive internal control. The synthesized RNA has a 22-base deletion with respect to the wild-type sequence. PCR products were quantitated by densitometric analysis of a digitalized image of the ethidium bromide stained gel. The non-radioactive method was evaluated in reconstruction experiments. RNA synthesis in FIV-infected feline thymocytes correlated well with the amount of viral p24 antigen produced. Viral RNA concentrations in the plasma of two cats experimentally infected with FIV strain UT113 were followed for 32 weeks; peak copy numbers (2.3 x 10(4) and 1.3 x 10(4) per ml, respectively) were reached 11 weeks after subcutaneous injection of ten 50% cat infectious doses. With rising antibody titers against FIV-gag and FIV-env gene products, the amount of FIV RNA in plasma decreased. Nine asymptomatic cats that had been experimentally infected 3.5 to 4.5 years earlier had copy numbers between 5.6 x 10(3) and 4.3 x 10(4) per ml. This quantitative competitive RT-PCR will be useful to study the pathogenesis of the FIV infection, to evaluate the effectiveness of vaccines and to monitor antiviral and immunomodulating drugs.

Asunto(s)

Virus de la Inmunodeficiencia Felina/aislamiento & purificación , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Secuencia de Bases , Enfermedades de los Gatos/virología , Gatos , Células Cultivadas , Cartilla de ADN , Síndrome de Inmunodeficiencia Adquirida del Felino/virología , Infecciones por Lentivirus/veterinaria , Infecciones por Lentivirus/virología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/aislamiento & purificación , Timo/citología , Timo/virología , Transcripción Genética

RESUMEN

The antiviral efficacy of acyclic nucleoside phosphonates, including 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine [(R)-PMPDAP] against feline immunodeficiency virus (FIV) infection was determined. (R)-PMPDAP showed the highest selectivity index (> 2,000) in vitro. Treatment of experimentally FIV-infected asymptomatic cats with PMEA or (R)-PMPDAP had no effect on the CD4+/CD8+ ratio. However, mean plasma viral RNA concentrations decreased significantly in the (R)-PMPDAP-treated cats. Our data show that, in comparison to PMEA, (R)-PMPDAP is a more potent and less toxic inhibitor of FIV replication both in vitro and in vivo.

Asunto(s)

Adenina/análogos & derivados , Antivirales/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Felino/tratamiento farmacológico , Virus de la Inmunodeficiencia Felina , Compuestos Organofosforados/uso terapéutico , Adenina/farmacocinética , Adenina/uso terapéutico , Animales , Antivirales/farmacocinética , Gatos , Síndrome de Inmunodeficiencia Adquirida del Felino/metabolismo , Técnicas In Vitro , Compuestos Organofosforados/farmacocinética , ARN Viral/biosíntesis , Linfocitos T/virología , Replicación Viral/efectos de los fármacos

Asunto(s)

Gatos/genética , Interleucina-4/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN/química , ADN Complementario/genética , Ciervos , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Porcinos

Asunto(s)

Gatos/genética , Interferón gamma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Perros , Genes , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido