RESUMEN
The objective of this study was to perform a comprehensive risk assessment on infectious disease transmission in the system of in vitro embryo production via somatic cell nucleus transfer (SCNT) technology using bovine viral diarrhea virus (BVDV) as a model. The risks of BVDV transmission in each step of the SCNT embryo production procedure, from donor cells to preimplantation SCNT embryo culture, were carefully examined using a sensitive real-time polymerase chain reaction assay. The identified primary source of BVDV transmission in SCNT embryo production was donor cell infection, most likely caused by contaminated fetal bovine serum in the culture medium. The risk of disease transmission through contaminated oocytes from an abattoir was relatively low, and it can be greatly minimized by cumulus cell removal and adequate oocyte washing procedures. Of the 200 cumulus-oocyte complexes (COCs) and more than 1500 cumulus cell-free oocyte (CFO) samples collected from multiple sources over a course of 7 months, only 2.5% of the COCs were BVDV positive, and all of the CFOs (100%) were BVDV negative. To evaluate the risk of BVDV introduction during in vitro SCNT embryo culture, 324 SCNT embryos were produced from 18 different cell lines using oocytes from 26 different batches collected over a course of 9 months. The embryos were cultured in vitro for 7 days and then tested for BVDV. All of the 324 SCNT embryos (100%) were negative, indicating that the embryo culture system is virtually risk-free for BVDV transmission. Based on these results, a standard operational protocol (SOP) for SCNT embryo production was proposed to greatly minimize the risk of BVDV transmission through the SCNT embryo production system. This SOP could be a starting point to produce a SCNT system that is virtually risk-free for disease transmission in general.
Asunto(s)
Diarrea Mucosa Bovina Viral/transmisión , Bovinos/virología , Clonación de Organismos/veterinaria , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Técnicas de Transferencia Nuclear/veterinaria , Animales , Blastocisto/virología , Bovinos/embriología , Línea Celular , Medios de Cultivo , Virus de la Diarrea Viral Bovina/genética , Técnicas de Cultivo de Embriones/veterinaria , Femenino , Sangre Fetal/virología , Fibroblastos/ultraestructura , Fibroblastos/virología , Oocitos/virología , Reacción en Cadena de la Polimerasa , ARN Viral/química , Medición de Riesgo , Alineación de SecuenciaRESUMEN
The objective was to develop a method to accurately and efficiently detect minute amounts of bovine viral diarrhea virus (BVDV) associated with a single embryo. There are two major challenges for BVDV detection in a single embryo: the test sensitivity and the efficiency of viral molecule recovery. These become even more critical when attempts are made to detect BVDV infections that occurred naturally, not through artificial exposure of the embryos to high affinity BVDV strains. We have developed a one-step sample preparation method that has increased the viral molecule recovery rate compared to the standard RNA isolation procedure by 7-100-fold. Instead of using the traditional virus exposure approach, we generated BVDV positive embryos via somatic cell nuclear transfer (SCNT) technology using BVDV positive donor cells. By combining the highly efficient sample preparation procedure with a sensitive one-step, real-time PCR system, we have developed a sensitive test that allows detection of as low as two copies of BVDV in a single embryo. This method will allow systematic risk assessment for BVDV transmission during in vitro embryo production via IVF or SCNT procedures.
