Nikolaus Osterrieder

Characterization of the Gene Encoding the A-Type Inclusion Body Protein of Mousepox Virus

The gene of the mousepox virus strain MP-1 coding for the protein of A-type inclusions was identified and sequenced. The gene maps in theHindIII-A fragment at the same position as the cowpoxvirus Brighton 160 kD gene (1) and the closely related 94 kD LS gene of vaccinia virus strain Western Reserve (2,3). The encoded protein consists of 1049 amino acids with a predicted molecular weight of 122.5 kD, which corresponds well to the apparent molecular weight of 130 kD estimated after polyacrylamide gel electrophoresis and Western blotting. The protein is a characteristic feature of mousepox virus and could be demonstrated for five independently isolated strains. The MP-1 ATI protein displays a similarity of 82.3% to the cowpox virus Brighton 160 kD protein, the first 340 amino acids being almost identical. However, two major deletions of 55 and 92 amino acids were observed from amino acid positions 671 to 732 and 794 to 885, respectively. Furthermore, the C-terminal residues (position 998–1049 of the MP-1 sequence) did not match at satisfactory levels with cowpox nor with vaccinia virus sequences. According to Western blot and protein alignment data as well as hydropathy predictions, the 130 and 160 kD proteins closely resemble each other, indicating similar structure and function.

A touchdown PCR for the differentiation of equine herpesvirus type 1 (EHV-1) field strains from the modified live vaccine strain RacH

More than 50 reference strains and field isolates of equine herpesvirus type 1 (EHV-1) were examined by a touchdown PCR. Primers for specific amplification of EHV-1 DNA were chosen from the terminal and internal repeat regions of the EHV-1 genome where the high-passaged live vaccine strain RacH displays symmetric 850 bp deletions. The positive strand and one negative strand primer were designed to encompass the deletions present in RacH, and the second negative strand primer was designed to hybridize within these deletions. Discrimination between field isolates and the vaccine strain was achieved by the generation of amplification products of different size: In all EHV-1 reference strains and field isolates, a 495 bp DNA fragment was amplified specifically, whereas a 310 bp fragment was amplified when DNA of the vaccine strain RacH was used as a template. PCR amplification was only obtained in the presence of 8-10% dimethylsulfoxide and when the primer annealing temperatures were decreased stepwise from 72 degrees C to 60 degrees C. Under these conditions as little as 100 fg template DNA, corresponding to about 100 genome equivalents, could be detected. The PCR assay allows fast and sensitive discrimination of the modified live vaccine strain RacH from field strains of EHV-1 since it is applicable to viral DNA extracted from organ samples and paraffin-embedded tissues. It may thus be helpful for examining the potential involvement of the RacH live vaccine strain in abortions of vaccinated mares.