RESUMEN
Bovine leukemia virus (BLV) infects cattle worldwide and causes B-cell lymphoma in cattle. BLV has been identified in human breast and lung cancer and in blood, but the association of BLV and human cancer is controversial. In this study, we investigated the existence of BLV in 145 Japanese human blood cell lines and 54 human cancer cell lines, using a new highly sensitive PCR assay that can amplify even one copy of BLV using LTR primers different from those in previous studies on BLV provirus in breast cancer. All samples were found negative for BLV provirus, suggesting that BLV is unlikely to infect humans.
Asunto(s)
Células Sanguíneas/virología , Línea Celular Tumoral/virología , Virus de la Leucemia Bovina/aislamiento & purificación , Zoonosis/diagnóstico , Adulto , Anciano , Animales , Células Sanguíneas/citología , Línea Celular , Línea Celular Tumoral/citología , Femenino , Humanos , Japón , Virus de la Leucemia Bovina/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Secuencias Repetidas Terminales , Adulto Joven , Zoonosis/virologíaRESUMEN
Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. Because BLV infection can remain clinically silent, the proviral load is an important index for estimating disease progression. CoCoMo-qPCR, an assay developed to estimate BLV proviral load, allows the highly sensitive detection of BLV originating in different countries. Here, we developed a modified version of the CoCoMo-qPCR assay, the "BLV-CoCoMo-qPCR-2" assay, which uses optimized degenerate primers. We also constructed a new plasmid standard. Finally, we used both assays to examine DNA samples from BLV-infected cattle and compared the results.
Asunto(s)
Leucosis Bovina Enzoótica/diagnóstico , Virus de la Leucemia Bovina/genética , Virus de la Leucemia Bovina/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Sondas de Oligonucleótidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Carga Viral/métodos , Animales , Bovinos , Leucosis Bovina Enzoótica/virología , Técnicas de Diagnóstico Molecular/normas , Plásmidos , Provirus/genética , Provirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Medicina Veterinaria/métodos , Medicina Veterinaria/normas , Carga Viral/normasRESUMEN
A VeraCode-allele-specific primer extension (ASPE) method was applied to the detection and genotyping of human papillomavirus (HPV)-DNA. Oligonucleotide primers containing HPV-type-specific L1 sequences were annealed to HPV-DNA amplified by PGMY-PCR, followed by ASPE to label the DNA with biotinylated nucleotides. The labeled DNA was captured by VeraCode beads through hybridization, stained with a streptavidin-conjugated fluorophore, and detected by an Illumina BeadXpress® reader. By using this system, 16 clinically important HPV types (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were correctly genotyped in a multiplex format. The VeraCode-ASPE genotyping of clinical DNA samples yielded identical results with those obtained by validated PGMY-reverse blot hybridization assay, providing a new platform for high-throughput genotyping required for HPV epidemiological surveys.
Asunto(s)
Alphapapillomavirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Infecciones por Papillomavirus/virología , Alelos , Alphapapillomavirus/clasificación , Alphapapillomavirus/genética , Cartilla de ADN/genética , Femenino , Genotipo , Humanos , Reacción en Cadena de la Polimerasa Multiplex/instrumentación , Infecciones por Papillomavirus/diagnósticoRESUMEN
Bovine leukemia virus (BLV) induces enzootic bovine leukosis, which is the most common neoplastic disease in cattle. Sero-epidemiological studies show that BLV infection occurs worldwide. Direct contact between infected and uninfected cattle is thought to be one of the risk factors for BLV transmission. Contact transmission occurs via a mixture of natural sources, blood, and exudates. To confirm that BLV provirus is detectable in these samples, matched blood, nasal secretion, and saliva samples were collected from 50 cattle, and genomic DNA was extracted. BLV-CoCoMo-qPCR-2, an assay developed for the highly sensitive detection of BLV, was then used to measure the proviral load in blood (n=50), nasal secretions (n=48), and saliva (n=47) samples. The results showed that 35 blood samples, 14 nasal secretion samples, and 6 saliva samples were positive for the BLV provirus. Matched blood samples from cattle that were positive for the BLV provirus (either in nasal secretion or saliva samples) were also positive in their blood. The proviral load in the positive blood samples was >14,000 (copies/1×10(5) cells). Thus, even though the proviral load in the nasal secretion and saliva samples was much lower (<380 copies/1×10(5) cells) than that in the peripheral blood, prolonged direct contact between infected and healthy cattle may be considered as a risk factor for BLV transmission.