Bioluminescent detection probe for tick-borne encephalitis virus immunoassay.

To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity.

GAG-binding variants of tick-borne encephalitis virus.

Previously different authors described various flavivirus mutants with high affinity to cell glycosaminoglycans and low neuroinvasiveness in mice that were obtained consequently passages in cell cultures or in ticks. In present study the analysis of TBEV isolates has shown existence of GAG-binding variants in natural virus population. Affinity to GAG has been evaluated by sorption on heparin-Sepharose.

Reverse transcription, amplification and sequencing of poliovirus RNA by Taq DNA polymerase.

A model for virion RNA of the poliomyelitis virus, which does not pass the stage of DNA copies during biogenesis, demonstrates that Taq DNA polymerase is capable of synthesizing 960-bp cDNA with the specific primer. When comparing the nucleotide sequence of the starting virion RNA and recombinant DNAs, isolated from several independent clones, copying and amplification of virion RNA appear accurate (one substitution per 960 bp). A comparison of Taq and Tth DNA polymerases in RT/PCR indicates that the sensitivity of Taq polymerase seems to be two orders of magnitude higher than that of Tth polymerase.

Effect of the multiplicity of infection on synthesis of the tick-borne encephalitis virus-specified proteins during a single reproduction cycle.

The rate of the synthesis of tick-borne encephalitis (TBE) virus-specified proteins at high multiplicity of infection (MOI of 100 PFU per cell) was the highest by 8-14 hr post-infection (p.i.) as compared to the lower MOI of 4 PFU per cell (14 to 17 hr p.

A tentative model of formation of structural proteins of tick-borne encephalitis virus (flavivirus).

The time course of tick-borne encephalitis virus cell-free protein synthesis was studied by using either [35S]-methionine or formyl[35S]methionyl-tRNAMet/f as substrates, and the [35S]methionine-labelled products were compared by fingerprinting tryptic peptides. An intermediate in the protein processing, the polypeptide doublet p36/33, was characterized and a tentative model for flavivirus structural protein synthesis and processing was proposed.