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The computer predicted secondary structures from the 5'-NC genome region of four border disease virus (BDV) strains collected from sheep in England and Scotland over a period 1976-1986 were prepared. The FOLD program from GCG sequence analysis software package was used for the analysis of a 243 bp RNA fragment. Two typical shapes of secondary structures were observed which contained multiple imperfect stem-loop motifs. The shape of those structures exactly fit with the grouping of BDV strains to two phylogenetic groups. Secondary structures are typical only of BDV strains and they are different from the structures prepared for NADL (BVDV) and Alfort (CSFV) strains.

The development of PCR assays for detection of BHV-1, BRSV, BVDV and another pestiviruses is summarized. A polymerase chain reaction assay based on primers selected from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size (468 bp) was amplified using DNA from herpesviruses isolated from reindeer, red deer and goat. The PCR assay was able to detect virus in nasal swabs 1-14 days after experimental infection of cattle and there was a good correlation when PCR was compared to virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful. PCR detecting a broad range of BVDV, BDV and HCV was developed. Of six sets of primers selected from different parts of the pestivirus genome the best results were provided by a pair 324/326 from the highly conserved 5'-non-coding region which gave an amplification with all 129 isolates tested. This panel consisted of 79 isolates from cattle, 33 from pigs and 17 from sheep. Differentiation between viruses was achieved by cleavage of the PCR-amplified products (288 bp) with the restriction endonucleases AvaI and BglI. The BVDV products were cleaved by AvaI, HCV by BglI and AvaI. Both enzymes, AvaI and BglI, did not cut the BDV products. A nested polymerase chain reaction assay was developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein. The sensitivity of PCR assay was 0.1 TCID50. No cross reaction was observed with nine heterologous respiratory viruses. PCR products of bovine and human RSV strains were discriminated using endonuclease ScaI, which specifically cleaved products of BRSV. PCR assay detected BRSV in nasal swabs collected from cattle in the acute stage of respiratory disease. In vitro amplification detected 31 positive samples of 35 while immunofluorescence only 23 samples.

DNA in-vitro amplification when a PCR (polymerase chain reaction) method is used (Saiki et al., 1985) provides for a simple technique of marked amplification of a selected DNA fragment. The length of a DNA amplified fragment is determined by two synthetic primers which spontaneously (at an appropriate temperature) hybridize with the opposite ends of antiparallelly oriented strains of denatured DNA. The enzyme Taq polymerase completes the synthetisation of new DNA strains from the primers. Repetition of these cycles (denaturing, primer bonds, DNA synthesis) enhances the DNA amplification of a defined strain length to such an extent that is possible to prove this process by e.g. electrophoretic analysis. For the purposes of a proof of BVD-MD genome in cattle the fragment 315 bp was chosen from the virus-coding gene gp 80. The primers P1-5'-GTAGGTAGAGTGAAACCCGG-3' and P2-5'-CGGGACCTGGACTTCATAGC-3' (Hertig et al., 1991) determined the length of the amplified fragment. Virus RNA was isolated from the infectious BVD-MD-containing medium (Ph strain) using a phenol-chloroform (1:1) mixture, and before amplification it was transcribed to cDNA in the P2 presence by the effect of AMV reverse transcriptase. cDNA without isolation from the transcription reaction mixture was directly used for PCR. DNA was denatured at 94 degrees C for 10 minutes before the outset of amplification. These reaction conditions are suitable for the PCR method: P1 and P2 primer concentrations per 100 microliters reaction solution-1 microM, dNTP-100 microM, 2-4 U Taq polymerase, 25-35 amplification cycles with the temperature regime: 94 degrees C/l min, 56 degrees C/l min, 73 degrees C/l min, and prolonged incubation 73 degrees C/7 min in the last cycle. Proof of the amplified product 315 bp DNA-electrophoretic analysis of 1.5 to 2% agarose gel in TAE buffer and ethidium bromide staining of gel are suitable. The introduced PCR method gives opportunities for innovations of BVD-MD diagnosis in cattle on the basis of virus genome proof without any cell cultivation need.

With the development of molecular biology in the 1980s methods of microorganism detection start to innovate. One of the main advantages of the molecular-genetic methods, namely hybridization of nucleic acids and PCR methods, is the detection of genome of microorganism without the need for cellular cultivation. To detect BHV-1 (etiological agens IBR-IPV) the dot-blot hybridization method on nitrocellulose filters was used together with different types of DNA probes (two-fiber recombinant plasmids, one-fiber recombinant phages M 13 and 40 bp synthetic oligonucleotide). Genome DNA BHV-1 was isolated from samples (virions, infested cells, nasal smears and secretions by phenol extraction). The highest sensitivity of detection was achieved with 32P-pUR-1 probe (1.8kb random EcoRI-Hind III fragment ligated into plasmid pUC 9) which detected genome BHV-1 in 5 x 10(3) infested MDBK cells. This probe did not respond with herpetic viruses BHV-2, BHV-3, BHV-4 and the virus of Aujeszky's disease. The quality of pUR-1 probe was further tested for IBR diagnostics in animals experimentally infested with the virus BHV-1 (intranasal infection). BHV-1 could be detected in nasal smears and secretions in experimentally infested calves as early as on the first day following infection, while the agens amount reached its peak on the days 2-3 and on the days 6-7 the occurrence of virus fell markedly. When digoxigenin-pUR-1, i.e. non-radioactively marked probe, the virus presence was confirmed only on the days 2-3, in the time of the highest occurrence of infection agens. To detect the virus through the dot-blot hybridization nasal secretions were confirmed as better compared with nasal smears. The technology of virus isolation on cell cultures confirmed also the occurrence of agens as soon as on the first day from infection, with maximum on the days 2-5, but much more reliably it detected the virus on the days observed from the day 3 and their peak was obtained on the day 6 from infection. Experiments, comparing classical methods of IBR diagnostics (detection of specific antibodies, the method of isolation on cellular cultures) with the dot-blot hybridization using the samples obtained from farms with natural occurrence of IBM, are under progress.

For the construction of the cDNA library total cellular RNA was separated from bovine leucocytes induced with NDV (inductor of interferons) for five hours. Guanidinisothiocyanate and ultracentrifugation in the gradient of CsCl were used for the separation of the total RNA (Chirgwin et al., 1979). Bands 18S and 28S were detected in the samples of RNA by electrophoresis in an MOPS buffer (Fig. 1). mRNA was separated from the mixture of RNA via affinity chromatography on oligo(dT)-cellulose (Aviv and Leder, 1972). The function quality of mRNA BoIFN-alpha was verified by microinjection into the oocytes of Xenopus laevis using a microinjector of our own construction (Fig. 2). The microinjector was calibrated by injection of a radioactive 51Cr solution (Fig. 3). It was found out on the basis of CPE inhibition measurements that the injected oocytes synthesized 320-640 U/ml BoIFN-alpha (16-32 U/50 microliters; Tab. I).