ABSTRACT
Prions are misfolded proteins that accumulate within the brain in association with a rare group of fatal and infectious neurological disorders in humans and animals. A current challenge to research is a lack of in vitro model systems that are compatible with a wide range of prion strains, reproduce prion toxicity, and are amenable to genetic manipulations. In an attempt to address this need, here we produced stable cell lines that overexpress different versions of PrPC through lentiviral transduction of immortalized human neural progenitor cells (ReN VM). Differentiated cultures made from the neural progenitor cell lines overexpressed PrPC within 3D spheroid-like structures of TUBB3+ neurons and we observed evidence that PrPC modulates formation of these structures, consistent with PrPC's role in neurogenesis. However, through repeated measurements of amyloid seeding activity in 6-week time course experiments, we failed to observe any evidence of prion replication within the differentiated ReN cultures following challenge with four prion isolates (human sCJD subtypes MM1 and VV2, and rodent adapted scrapie strains RML and 263K). We attributed amyloid seeding activity detected within the cultures to residual inoculum and concluded that PrPC overexpression was insufficient to confer permissiveness of ReN cultures to prion infection. While our ReN cell prion infection model was unsuccessful, additional efforts to develop cellular models of human prion disease are highly warranted.
Subject(s)
Creutzfeldt-Jakob Syndrome , Prion Diseases , Prions , Animals , Humans , Prions/metabolism , Creutzfeldt-Jakob Syndrome/genetics , Prion Diseases/metabolism , Cell Line , Stem Cells/metabolismABSTRACT
Foot-and-mouth disease (FMD) and vesicular stomatitis (VS) cause such similar clinical signs and lesions that laboratory tests are required to distinguish between infections caused by each virus. Using mouse anti-foot-and-mouth disease virus (FMDV) 3B monoclonal or polyclonal anti-vesicular stomatitis virus-New Jersey (VSV-NJ) antibodies and recombinant FMDV 3ABC or VSV-NJ glycoprotein (G) antigens coated to MagPlex beads, competitive Luminex immunoassays (cLIAs) were developed for FMDV and VSV-NJ, respectively. The cLIAs successfully detected antibodies to FMDV 3ABC and VSV-NJ G in sera from infected animals. The diagnostic sensitivity and specificity were 93% and 98%, respectively for FMDV and 93% and 95.4%, respectively for VSV-NJ. These cLIAs are potential alternatives for competitive enzyme-linked immunosorbent assays (cELISAs) and provide the opportunity for multiplexing to reduce time and the amount of serum required for testing.
La fièvre aphteuse (FA) et la stomatite vésiculaire (SV) causent des signes cliniques et des lésions tellement similaires que des tests de laboratoire sont requis afin de distinguer entre les infections causées par chaque virus. En utilisant un anticorps monoclonal 3B de souris anti-virus de la fièvre aphteuse (VFA) ou un anticorps polyclonal anti-virus de stomatite vésiculaire-New Jersey (VSV-NJ) et des billes MagPlex enduites d'antigènes de VFA recombinant 3ABC ou de glycoprotéine G de VSV-NJ, des immuno-essais Luminex compétitifs (cLIAs) furent développés pour le VFA et le VSV-NJ, respectivement. Les cLIAs ont détecté avec succès des anticorps contre VFA 3ABC et VSV-NJ G dans le sérum d'animaux infectés. La sensibilité et spécificité diagnostiques étaient de 93 % et 98 %, respectivement pour le VFA et de 93 % et 95,4 %, respectivement pour le VSV-NJ. Ces cLIAs sont des alternatives potentielles pour les épreuves ELISA compétitives et fournissent l'opportunité de multiplexer afin de réduire le temps et la quantité de sérum requis pour les tests.(Traduit par Docteur Serge Messier).
Subject(s)
Antibodies, Viral/blood , Foot-and-Mouth Disease/diagnosis , Immunoassay/veterinary , Vesicular Stomatitis/diagnosis , Animals , Diagnosis, Differential , Foot-and-Mouth Disease/immunology , Foot-and-Mouth Disease Virus/immunology , Sensitivity and Specificity , Species Specificity , Vesicular Stomatitis/immunology , Vesicular stomatitis Indiana virus/immunologyABSTRACT
BACKGROUND: Foot-and-mouth disease (FMD) is an economically devastating disease that severely limits international trade of animals. Of the seven FMD virus (FMDV) serotypes, serotype A is one of the most widespread cross the world. Currently antibodies to FMDV are detected in animals using the virus neutralization test (VNT) and the enzyme-linked immunosorbent assay (ELISA). The VNT is laborious, time-consuming and reliant on live virus and cell cultures, while ELISA has the advantage of using inactivated antigens and often provides more reproducible results. The aim of this study was to develop a reliable and rapid competitive ELISA (cELISA) for the detection of antibodies to FMDV serotype A (FMDV/A). RESULTS: A panel of FMDV/A specific monoclonal antibodies (mAbs) was generated and their ability to compete with a polyclonal serum from FMDV/A-infected cattle was examined. Two mAbs inhibited the binding of a polyclonal serum to FMDV/A viruses. The binding epitopes of each were determined as conformational and located on the VP2 viral capsid protein. The FMDV/A cELISA was developed using these two mAbs and FMDV/A inactivated virus as antigen. The diagnostic specificity and sensitivity were 99.7 and 99.3% (98.5-100%) respectively, based on a predetermined cut-off of 50% inhibition. When analysing sera from animals experimentally infected with FMDV/A, the cELISA detected antibodies from 5-days post infection (dpi) and remained positive for at least 21-28 days post infection. Comparison based on the Kappa coefficient showed strong agreement (90-94%) between cELISA and VNT. CONCLUSION: The cELISA results are comparable to the VNT for antibody detection making it a simple and reliable test to detect antibodies against FMDV/A.