SUMMARYA major Mycoplasma gallisepticum polypeptide of 64 kDa (p64) was characterized using two distinct monoclonal antibodies (MAbs), MAb KI produced in our laboratory and MAb MyG 001 produced by Avakian & Ley (1993). The p64 antigen was shown to be a lipoprotein in a radioimmunoprecipitation assay using [ 3 H] palmitic acid-labelled M. gallisepticum cultures. The two MAbs inhibited the growth of M. gallisepticum in liquid medium and reacted to two distinct epitopes on the same p64 antigen in competitive enzyme-linked immunosorbent and chemiluminescence Western immunoblot assays. MAb Kl inhibited haemagglutination of chicken and turkey erythrocytes whereas MAb MyG 001 did not. The results of our study indicate that p64 has two distinct epitopes involved in haemagglutination and growth inhibition of M. gallisepticum. MAb Kl also inhibited the attachment of the mycoplasma to TLT lymphoblastoid chicken B cell line, suggesting that p64 is a cytadhesin.

The purpose of this study was to develop a sensitive, rapid, and inexpensive immunofluorescence assay (IFA) using a recombinant porcine circovirus type 2 (PCV2) nucleocapsid protein for the serological detection of PCV2-specific antibodies in pig sera. The viral nucleocapsid protein encoded by the PCV2 ORF2 gene has recently been identified as the most immunoreactive viral protein that carries type-specific antigenic determinants. The ORF2 sequence of the IAF-2897 strain of PCV2 has been cloned into a pCEP5 eucaryotic expression vector under the control of the cytomegalovirus promoter, downstream of a polyhistidine sequence tag. The recombinant plasmid was used in transfection experiments with human epithelial kidney 293 cells that were further tested, and positive expression of the viral nucleocapsid protein was confirmed by IFA and Western blotting. Strong, specific fluorescence was observed in the nuclei of transfected cells. Test specificity to PCV2 was verified with several related infectious agents. Sensitivity was compared to that of standard IFA using PCV2-infected cells by evaluating the reactivities of 44 field serum samples from pigs on farms with a porcine population suffering from postweaning multisystemic wasting syndrome. The recombinant nucleocapsid-based test was able to detect 15 more positive-testing pigs than the PCV2-based IFA. Therefore, the relative sensitivity of the latter test was estimated at only 57.1% compared to that of the recombinant nucleocapsidbased test. The recombinant fusion protein has been purified by affinity chromatography and is being used to develop further sensitive serological tests.

Pigs exposed to GP5 protein of PRRSV by means of DNA immunization develop specific neutralizing and protecting antibodies. Herein, we report on the consequences of codon bias, and on the favorable outcome of the systematic replacement of native codons of PRRSV ORF5 gene with codons chosen to reflect more closely the codon preference of highly expressed mammalian genes. Therefore, a synthetic PRRSV ORF5 gene (synORF5) was constructed in which 134 nucleotide substitutions were made in comparison to wild-type gene (wtORF5), such that 59% (119) of wild-type codons were replaced with known preferable codons in mammalian cells. In vitro expression in mammalian cells of synORF5 was considerably increased comparatively to wtORF5, following infection with tetracycline inducible replication-defective human adenoviral vectors (hAdVs). After challenge inoculation, SPF pigs vaccinated twice with recombinant hAdV/synORF5 developed earlier and higher antibody titers, including virus neutralizing antibodies to GP5 than pigs vaccinated with hAdV/wtORF5. Data obtained from animal inoculation studies suggest direct correlation between expression levels of immunogenic structural viral proteins and immune response

No abstract

The 5(-terminal leader sequence of the equine arteritis virus (EAV) genome contains an open reading frame (ORF) with an AUG codon in a suboptimal context for initiation of protein synthesis. To investigate the significance of this intraleader ORF (ILO), an expression plasmid was generated carrying a DNA copy of the subgenomic mRNA7 behind a T7 promoter. Capped RNA transcribed from this construct was shown to direct, in an in vitro translation system, the synthesis of leader peptide as well as N protein. Site-directed mutations aimed to either optimize or weaken the sequence context of the ILO start codon affected leader peptide synthesis as predicted; no peptide was detected when the initiation codon was incapacitated. Translation of the downstream N gene was inversely affected by leader peptide production, consistent with a ribosomal leaky scanning mechanism. To investigate the role of the leader peptide in the EAV replication life cycle we generated, using an infectious EAV cDNA clone, two mutant viruses in one of which the ILO start codon was in an optimal Kozak context for translation initiation while in the other the codon was again incapacitated. Surprisingly, both mutant viruses were equally viable and exhibited similar phenotypes in BHK-21 cells. However, their replication kinetics and viral yields were reduced relative to that of the wild-type parental virus, as were their plaque sizes. Importantly, the mutations introduced into the viruses appeared to be rapidly and precisely repaired upon passaging. Already after one viral passage a significant fraction of the viruses had regained the wild-type sequence as well as its phenotype. The results demonstrate that EAV replication is not dependent on the synthesis of the intraleader peptide. Rather, the leader peptide does not seem to have any function in the EAV life cycle. As we discuss, the available data indicate that the ILO 5( nucleotide sequence per se, not its functioning in translation initiation, is of critical importance for EAV replication.