Two types of particles were isolated during purification of rotavirus. Dense (D) particles have a density of 1.38 in CsCl and exhibit spontaneously a fully active endogenous transcriptase. Light (L) particles (density of 1.36 in CsCl) need to be treated with chelating agents to show a polymerase activity. The activation process of L particles was studied under strictly controlled monovalent, divalent, and hydrogen ion concentrations. These experiments demonstrate that i) activation is not affected by the ionic strength ii) activation occurs only at a pH higher than 7.1 iii) a low concentration of chelating agent (40 muM EDTA) is sufficient to activate the enzyme. Treatment of particles with EGTA, which chelates selectively Ca2+, leads to unmasking even in the presence of magnesium, indicating that the concentration of free calcium ions plays a major role in the activation process. Various glycosidases, detergents, and chelating agents were tested in respect to unmasking properties. Of these compound only chelating agents turned out to be efficient. Following activation, two glycopeptides were solubilized. These glycopeptides have an apparent molecular weight of 34,000 and 31,000 daltons and react with concanavalin A. The role of Ca2+ upon the stability of virus particles, and the activation of the endogenous transcriptase in vitro and in the infected cells is discussed.

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Diarrheic feces from 21 calves were examined by electron microscopy and 16 contained particles morphologically similar to those of Breda virus. The particles were spherical or elongated, 60-270 nm in greatest dimension and had surface spikes 9-13 nm long. Convalescent serum from a human patient with Breda virus-associated diarrhea reacted with one of the bovine viruses by immune electron microscopy, suggesting a serological resemblance between human and bovine Breda-like viruses. Immune electron-microscopy and immunofluorescence demonstrated that isolates of bovine Breda virus from the U.S.A. were related to the French virus. One of the viruses had a density in sucrose solution of 1.16, similar to the value for Berne virus.

We have identified the binding site of monoclonal antibodies (MAbs) against the $2 subunit of the bovine coronavirus spike (S) glycoprotein. The location of this site was first investigated by using prokaryotic expression of DNA restriction fragments covering the entire S gene. The amino acid sequence containing the antibody binding site was shortened from 70 to 20 amino acids by digestion of plasmid DNA with exonuclease III, followed by sequencing of the smallest digestion product encoding an immunoreactive fusion protein. Finally we synthesized a set of nonapeptides covering the 20 amino acid sequence extending from the N-terminal residue of the $2 subunit (Ala 769 to Tyr 798). MAbs reacted mainly with six consecutive overlapping peptides with the sequence TTGYRFTN-FEPFTV. Polyclonal antibodies from hyperimmunized or convalescent animals reacted only with the recombinant proteins identified by MAbs, and the hyperimmune serum bound to the same set ofpeptides. This suggests that this highly conserved linear antigenic determinant corresponds to an immunodominant region. This region resembles both in location and immunodominance the linear determinant defined on the infectious bronchitis virus $2 subunit. The presence of similar regions in the N-terminal region of the $2 subunit of other coronaviruses is discussed.