RESUMO
Fetal bovine serum (FBS) used in cell culture may be contaminated with viruses, among them bovine viral diarrhea virus (BVDV) affecting the production of biological reagents and the results of diagnosis. The filtration process used in the preparation of commercial FBS abrogates most viral agents that may be present in raw FBS, but BVDV may pass through the filters because of its small size and its pleomorphism. While detection of bovine herpes virus-1 and parainfluenza-3 (PI-3) is determined by observation of the cytopathic effect, and also by hemadsorption in the case of PI-3, the most frequently isolated BVDV is non cytopathic, and infects cells without morphological alterations, inducing problems that arise after several cell generations. Batches of raw and processed FBS were analyzed. Frequencies of BVDV detection in raw serum in Argentina were similar to those published for USA. By conventional methods for BVDV detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell cultures maintained with high concentrations of the serum under study for at least 2 weeks, with detection of viral antigen by indirect immunofluorescence, the percentage of BVDV detection was 80%. This method shows that most lots of commercial FBS contain BVDV. RT-PCR allows faster detection of the viral genome, but it must be validated, as it does not show viral replication. To eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS is used in our laboratory.
Assuntos
Sangue/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Animais , Bovinos , Meios de CulturaRESUMO
Fetal bovine serum (FBS) used in cell culture may be contaminated with viruses, among them bovine viral diarrhea virus (BVDV) affecting the production of biological reagents and the results of diagnosis. The filtration process used in the preparation of commercial FBS abrogates most viral agents that may be present in raw FBS, but BVDV may pass through the filters because of its small size and its pleomorphism. While detection of bovine herpes virus-1 and parainfluenza-3 (PI-3) is determined by observation of the cytopathic effect, and also by hemadsorption in the case of PI-3, the most frequently isolated BVDV is non cytopathic, and infects cells without morphological alterations, inducing problems that arise after several cell generations. Batches of raw and processed FBS were analyzed. Frequencies of BVDV detection in raw serum in Argentina were similar to those published for USA. By conventional methods for BVDV detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell cultures maintained with high concentrations of the serum under study for at least 2 weeks, with detection of viral antigen by indirect immunofluorescence, the percentage of BVDV detection was 80. This method shows that most lots of commercial FBS contain BVDV. RT-PCR allows faster detection of the viral genome, but it must be validated, as it does not show viral replication. To eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS is used in our laboratory.(AU)
Assuntos
Animais , Bovinos , Sangue/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Meios de CulturaRESUMO
Fetal bovine serum (FBS) used in cell culture may be contaminated with viruses, among them bovine viral diarrhea virus (BVDV) affecting the production of biological reagents and the results of diagnosis. The filtration process used in the preparation of commercial FBS abrogates most viral agents that may be present in raw FBS, but BVDV may pass through the filters because of its small size and its pleomorphism. While detection of bovine herpes virus-1 and parainfluenza-3 (PI-3) is determined by observation of the cytopathic effect, and also by hemadsorption in the case of PI-3, the most frequently isolated BVDV is non cytopathic, and infects cells without morphological alterations, inducing problems that arise after several cell generations. Batches of raw and processed FBS were analyzed. Frequencies of BVDV detection in raw serum in Argentina were similar to those published for USA. By conventional methods for BVDV detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell cultures maintained with high concentrations of the serum under study for at least 2 weeks, with detection of viral antigen by indirect immunofluorescence, the percentage of BVDV detection was 80. This method shows that most lots of commercial FBS contain BVDV. RT-PCR allows faster detection of the viral genome, but it must be validated, as it does not show viral replication. To eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS is used in our laboratory.
Assuntos
Animais , Bovinos , Sangue , Vírus da Diarreia Viral Bovina/isolamento & purificação , Meios de CulturaRESUMO
Fetal bovine serum (FBS) used in cell culture may be contaminated with viruses, among them bovine viral diarrhea virus (BVDV) affecting the production of biological reagents and the results of diagnosis. The filtration process used in the preparation of commercial FBS abrogates most viral agents that may be present in raw FBS, but BVDV may pass through the filters because of its small size and its pleomorphism. While detection of bovine herpes virus-1 and parainfluenza-3 (PI-3) is determined by observation of the cytopathic effect, and also by hemadsorption in the case of PI-3, the most frequently isolated BVDV is non cytopathic, and infects cells without morphological alterations, inducing problems that arise after several cell generations. Batches of raw and processed FBS were analyzed. Frequencies of BVDV detection in raw serum in Argentina were similar to those published for USA. By conventional methods for BVDV detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell cultures maintained with high concentrations of the serum under study for at least 2 weeks, with detection of viral antigen by indirect immunofluorescence, the percentage of BVDV detection was 80
. This method shows that most lots of commercial FBS contain BVDV. RT-PCR allows faster detection of the viral genome, but it must be validated, as it does not show viral replication. To eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS is used in our laboratory.
RESUMO
Bovine Viral Diarrhea Virus (BVDV) is a pathogen of cattle, member of the family Flaviviridae, genus pestivirus, which also includes Classical Swine Fever Virus (CSFV, or hog cholera virus), and Border Disease Virus of sheep (BDV). It causes important economical losses associated mainly with reproductive failure. Pestiviruses are small enveloped viruses, with a diameter of about 40 nm. The nucleocapsid is probably icosahedral . The genome consists of a single stranded positive RNA, encoding approximately 430 kD of proteic product. Genetic expression consists of the synthesis of a polyprotein which is co- and post-translationally processed. According to its behavior "in vitro" two biotypes can be distinguished: non cytopathic (ncp) and cytopathic (cp), most probably derived from the ncp through mutations and/or recombination. BVDV is able to cross the placenta and infect the fetus, causing a variety of problems, from fetal death to the birth of a persistently infected (P) calf, according to the fetal age at the time of infection. PI animals are immunotolerant to the virus and shed it in all secretions. Only the ncp biotype has been isolated from PI animals. The superinfection of a PI animal with a cp strain causes mucosal disease, always fatal. Outbreaks of a severe, sometimes hemorrhagic disease, caused by ncp BVDV, have occurred in Canada and USA since 1993. Genomic and serological differences between the "traditional" strains and the viruses isolated from these outbreaks led to the division of BVDV in subtypes I and II, both including cp and ncp strains. Analyses of the non coding 5'-UTR zone of the genome of pestiviruses from different species (bovine, ovine, porcine) suggest that there are at least 3 genotypes within the genus. A new classification of these viruses, based on genomic sequence instead of species of origin, has been proposed. Genomic heterogeneity exists in the BVDV genome, which presents 3 hypervariable zones, 2 of them in the major neutralizing protein. In Argentina prevalence of BVDV antibodies in cattle population is 70%, and the prevalence of persistent infections is around 1%.