A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein.

Truncated NS3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved B-cell epitopes on this non-structural bovine viral diarrhoea virus (BVDV) protein. A goat anti-pestivirus antiserum, and a panel of anti-NS3 monoclonal antibodies, including the BVDV-1 specific antibody P1D8, were used to verify the presence or absence of the epitopes. Interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain of NS3. Expression of the B-cell epitopes was dependent on, but not within, a 57 amino acid sequence at the carboxy-terminal end of this protein, supporting observations that these conserved epitopes are conformational in nature. A comparison of deduced amino acid sequences of the helicase domain from BVDV-1, BVDV-2, BDV and CSFV isolates highlighted a single amino acid that appeared to be unique to P1D8-reactive BVDV-1 isolates. Site-directed mutagenesis studies confirmed that this amino acid is critical for the expression of the BVDV-1 specific NS3 epitope recognised by the P1D8 monoclonal antibody. Surprisingly, the amino acid was also important for an epitope recognised by two group-specific monoclonal antibodies, P1H11 and P4A11. Protein modelling studies, based on the structure of the hepatitis C NS3 helicase domain, indicated that this amino acid occupies a prominent position on the surface of the protein.

Mutational analysis of the NH2-terminal arms of the trp repressor indicates a multifunctional domain.

The NH2-terminal arms of the Escherichia coli trp repressor have been implicated in three functions: formation of repressor-operator complexes via association with non-operator DNA; stabilization of repressor oligomers bound to DNA; and oligomerization of the aporepressor in the absence of DNA. To begin to examine the structural aspects of the arms that are responsible for these varied activities, we generated an extensive set of deletion and substitution mutants and measured the activities of these mutants in vivo using reporter gene fusions. Deletion of any part of the arms resulted in a significant decrease in repressor activity at both the trp and the trpR operons. Positions 4, 5 and 6 were the most sensitive to missense changes. Most substitutions at these positions resulted in repressors with less than 5% of the activity of the wild-type trp repressor. A large percentage of the missense mutants were more active than the wild-type repressor in medium containing tryptophan and less active in medium without tryptophan. This phenotype can be explained in terms of altered oligomerization of both the repressor and the aporepressor. Also, nine super-repressor mutants, resulting from substitutions clustered at both ends of the arms, were found. Our results support the hypothesis that the NH2-terminal arm of the trp repressor is a multifunctional domain and reveal structural components likely to be involved in the various functions.

Insemination of cattle with semen from a bull transiently infected with pestivirus.

When 73 heifers (60 of which were seronegative to pestivirus) were inseminated with pestivirus-contaminated semen from a transiently infected bull, the conception rate to a single insemination was found to be normal (65 per cent). Only three animals became systemically infected, as determined by viraemia and seroconversion. Pestivirus was isolated from the reproductive tracts of two of these heifers when they were slaughtered 42 or 43 days after insemination. Although the initial incidence of infection was low, a cycle of secondary transmission occurred approximately 29 days after insemination, with a further eight heifers (all seronegative) becoming infected from one group of 11 seronegative and four seropositive animals.

The outcome of widespread use of semen from a bull persistently infected with pestivirus.

During the certification of the bulls at an artificial breeding centre for freedom from pestivirus infection, a single viraemic bull was identified, and further testing confirmed that it was persistently infected. The two-year-old bull was healthy and of similar bodyweight to its peers. Its semen was of normal quality on the basis of density, motility and morphological criteria. Approximately 600 doses of semen had been distributed for sire evaluation purposes to 97 dairy farms. An examination of the breeding records indicated a first service conception rate of 38 per cent. All but one of the 162 cows inseminated with the bull's semen were seropositive compared with 95 of 143 cows (66.4 per cent) inseminated with semen from other bulls. Virological studies of the 61 calves sired by the persistently infected bull revealed that two were persistently infected, but that the others were healthy and uninfected. It was concluded that the semen from this bull was a potential source of pestivirus infection for 'clean' herds.

Detection of hog cholera virus antigens in experimentally-infected pigs using an antigen-capture ELISA.

An antigen-capture ELISA was used to detect hog cholera virus (HCV) antigens in blood and tissues taken from pigs infected with 2 different strains of virus. Specific antigens were demonstrated in peripheral blood leucocytes (PBLs) and a wide range of tissue samples 4-6 days after infection of pigs with a moderate-high virulent HCV strain (Weybridge virus). Strong signal to noise (S/N) ratios were obtained in the ELISA for PBLs and lymphoid tissues such as spleen, tonsil and mesenteric lymph nodes at 5-7 days after infection with the Weybridge virus, S/N ratios varying between 8.1-19.7 for blood samples and 4.3-19.1 for spleen samples. High positive ELISA results were also obtained for duodenum and ileum samples (S/N ratios 10.3-18.6) taken from these pigs, reflecting severe pathological changes observed in the gut at post mortem. In contrast, the antigen-capture ELISA gave strong positive results for PBLs and spleen samples only at 7-9 days after infection of pigs with a low virulent strain of HCV (New South Wales virus). The ELISA S/N ratios averaged 9.5 for PBLs and 8.9 for spleen samples in these animals. Although virus isolation detected infection earlier in the infected pigs, the ELISA returned positive results on PBLs and spleen samples around the time all of the animals first showed typical signs of classical swine fever. The technique does not require tissue culture and takes less than 36 h to return a definitive result.