ABSTRACT
Cells from various tissues and species are able to bind to Theiler's virus strain DA and allow it to replicate to some extent. Meanwhile, permissiveness in vitro to BeAn strains has not been well investigated. In this paper, the BeAn 8386 virus was subjected to five passages in BHK-21 cells and showed a persistent profile. In order to follow the in vitro infection, real-time RT-PCR to detect the IRES, L* and 3A3B regions of the Theiler's virus genome was carried out in the first and last passages. In addition, the expression of L* protein was detected. These findings confirm the persistence of the virus in vitro, even in the absence of cytopathic effect (CPE).
Subject(s)
Gene Expression Regulation, Viral/physiology , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Theilovirus/classification , Theilovirus/genetics , Viral Proteins/metabolism , Animals , Cell Line , Cricetinae , Cytopathogenic Effect, Viral , Genetic Variation , Genome, Viral , Viral Proteins/genetics , Virus ReplicationABSTRACT
Monoclonal antibodies (MAbs) against human recombinant interferon beta (hrIFNbeta) were generated by genetic immunization (GI). In order to test two viral promoters frequently used in mammalian expression plasmid vectors, mice were inoculated four times by intramuscular injection, without adjuvant, with 100 microg of either pcDNA 3.1hrIFNbeta or pZeoSV2IFNbeta containing the entire human interferon beta gene and under the control of, respectively, human cytomegalovirus (HCMV) immediate-early promoter or early SV-40 enhancer/promoter. Only serum samples from mice immunized with pZeoSV2IFNbeta were positive to anti-hrIFNbeta. The spleens of the immunized mice were fused with myeloma Sp2/0 cells and the hybridoma clones generated screened by an in house enzyme-linked immunosorbent assay (ELISA). Fourteen MAbs were selected as reactive with hrIFNbeta. Western blot analysis was performed and only one recognized the 18 kDa isoform (non-glycosylated) of hrIFNbeta. All MAbs were subjected to antibody isotype characterization with a commercial ELISA and showed unusual profile with simultaneous expression of both IgM and IgG2a isotypes. This observation is further supported by RT-PCR amplification of the IgM CH4 domain using total RNA from hybridomas.
