Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from insect larvae.

BACKGROUND: There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in E. coli, yeast and baculovirus grown in insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV). FINDINGS: Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in insect larvae. Recognition of G21-465 by ß-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (Oncorhynchus mykiss) was demonstrated. CONCLUSIONS: Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies.

Measurement of the rates of synthesis of three components of ribosomes of Mycobacterium fortuitum: a theoretical approach to qRT-PCR experimentation.

BACKGROUND: Except for the ribosomal protein L12 (rplL), ribosomal proteins are present as one copy per ribosome; L12 (rplL) is unusual because it is present as four copies per ribosome. Thus, the strategies used by Mycobacterium fortuitum to regulate ribosomal protein synthesis were investigated, including evaluations of the rates of chain elongations of 16S rRNA, rplL and ribosomal protein S12 (rpsL). METHODOLOGY: RNA was isolated from cell cultures and cDNA was prepared. The numbers of cDNA copies of 16S rRNA, precursor-16S rRNA and transcripts of rpsL and rplL were quantified by qRT-PCR and then related to the rates of 16S rRNA, rpsL and rplL chain elongations by means of a mathematical framework for coupled transcription/translation. PRINCIPAL FINDINGS: The rates of synthesis of 16S rRNA, rpsL and rplL respectively were found to be approximately 50 x 10(3) nucleotides h(-1), 1.6 x 10(3) amino acid residues h(-1) and 3.4 x 10(3) amino acid residues h(-1). The number of transcripts of rplL was approximately twice that of rpsL. These data account for the presence of one copy of rpsL and four copies of rplL per ribosome, and reveal that the rate of M. fortuitum ribosome synthesis was closer to that of M. tuberculosis than to E. coli. Except for rplJ, the elongation rate obtained for rpsL was inferred to be appropriate for all other proteins present as one copy per ribosome. SIGNIFICANCE: The results obtained provide the basis for a comprehensive view of the kinetics of ribosome synthesis, and of the ways that bacterial cells utilize genes encoding ribosomal proteins. The methodology also applies to proteins involved in transcription, energy generation and to bacterial proteins in general. The method proposed for measuring the fidelity of cDNA preparations is intrinsically much more sensitive than procedures that measure the integrity of 16S rRNA.

Transcriptional analysis of Mycobacterium fortuitum cultures upon hydrogen peroxide treatment using the novel standard rrnA-P1.

BACKGROUND: The ability of an intracellular pathogen to establish infection depends on the capacity of the organism to survive and replicate inside the host. Mycobacterium fortuitum is a bacteria that contains genes involved in the detoxification of the oxygen reactive species such as those produced by the host during the infection. In this work, we investigate the effects of hydrogen peroxide on the transcription and expression of these genes by developing a real time quantitative PCR technique (qRT-PCR) using the ribosomal promoter region (rrnA-P1) as reference product for quantification of the mRNA levels. RESULTS: M. fortuitum cultures were treated with different hydrogen peroxide concentrations (0.02 to 20 mM) during several periods of time (30 to 120 minutes). The activity of the enzymes KatGII and SodA, and the transcription of corresponding genes were evaluated. The transcriptional regulator furAII gene was also studied. The ribosomal promoter region rrnA-P1 was validated as referential product under the stress conditions checked by qRT-PCR. Minor changes were observed under the conditions tested except when bacteria were incubated in the presence of 20 mM hydrogen peroxide. Under those conditions, the levels of transcription of the three genes under study increased at 30 minutes of treatment. The viability of the bacteria was not influenced under the conditions tested. CONCLUSION: In this work, we have quantified transcriptional responses to stress suggesting that, the opportunistic pathogen M. fortuitum is more resistant and differs in behaviour in the presence of hydrogen peroxide, when compared to the major pathogen Mycobacterium tuberculosis and the saprophyte Mycobacterium smegmatis. Besides, we demonstrate the mycobacterial non-coding region rrnA-P1 to be a suitable reference product in the analysis of qRT-PCR transcriptional data of M. fortuitum.