Graham J. Cox

Polynucleotide vaccines in animals : enhancing and modulating responses

We immunized cattle, the natural host for bovine herpesvirus 1 (BHV-1), with a polynucleotide vaccine encoding BHV-1 glycoprotein D. These cattle trials clearly indicate that large species can be immunized with polynucleotide vaccines. Recently, using a murine model, we demonstrated that: the cellular compartment to which the expressed antigen is delivered determines the type of immune response (type 1 or type 2), and that the magnitude and direction of the immune response can be modulated by coadministration of plasmid encoded cytokines and antigen. Finally, we demonstrated that immunization of mice with a polynucleotide vaccine encoding BHV-1 gD could circumvent preexisting passively transferred, gD specific, polyclonal antisera and lead to the development of an active immune response.

Primary structure of the S peplomer gene of bovine coronavirus and surface expression in insect cells

The nucleotide sequence of the S peplomer gene of bovine coronavirus (BCV) has been determined. A single open reading frame of 4089 nucleotides encodes a polypeptide of 150K with 20 potential sites for addition of N-linked oligosaccharides. Expression of the cloned BCV S gene by a recombinant of Autographa californica nuclear polyhedrosis virus resulted in production of a 180K glycosylated polypeptide which was transported to the surface of the cell. Comparison of the BCV S gene with the analogous genes of murine hepatitis viruses shows that the BCV S polypeptide contains a unique domain of 138 amino acids not present in murine hepatitis virus strain JHM, but which has a partially homologous counterpart in strain A59. This domain accounts for most of the differences in size of the S gene products of these coronaviruses.

Bovine coronavirus nonstructural protein ns2 is a phosphoprotein

Abstract To investigate the nature of the bovine coronavirus (BCV) ns2 protein, the gene encoding this protein was cloned and was expressed as a β-galactosidase fusion protein. Antiserum raised against this protein reacted specifically with BCV-infected fixed cells in indirect immunofluorescence microscopy and precipitated an in vitro synthesized product approximately 32-kDa in molecular weight and an equivalent protein from BCV-infected cells. The synthesis of ns2 was found to be similar to the structural proteins of BCV and pulse-chase experiments indicated that ns2 protein was stable and that it accumulated in BCV-infected cells. Synthesis of ns2 in the presence of [31P] orthophosphate revealed that it is a phosphoprotein. Phosphoamino acid analysis confirmed the phosphorylated nature of ns2 and identified serine and threonine as its phosphorylated amino acid residues. This is the first demonstration of a phosphorylated nonstructural protein in coronavirus-infected cells.

Zinc-Binding of the Cysteine-Rich Domain Encoded in the Open Reading Frame 1B of the RNA Polymerase Gene of Coronavirus

We cloned and sequenced the second open reading frame of the RNA polymerase gene, ORF1b, of bovine coronavirus. In the region representing nucleotide positions 4919-5677 upstream from the initiation codon of the 32K non-structural protein gene, we identified two putative functional domains. One of these domains contained four leucine residues repeated exactly in every seventh position, and the other domain represented a cluster of cysteine and histidine residues. The DNA sequence representing these domains was cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. A high level expression of the cysteine-rich domain was achieved as a fusion protein when the bacterial culture was induced with IPTG. In a solid phase zinc binding assay using the recombinant fusion protein, we found that the protein containing the cysteine-rich domain was able to bind to radioactive zinc in vitro, demonstrating that the polypeptide encoded by the ORF1b of coronavirus is a zinc-binding protein.

The Haemagglutinin of Bovine Coronavirus Exhibits Significant Similarity to the Haemagglutinin of Type C Influenza Virus

Coronaviruses are large enveloped RNA viruses containing a single-stranded genome complexed with a phosphorylated nucleocapsid protein which is surrounded by an envelope derived from intracellular membranes. The envelope contains two or three species of glycoproteins. The largest, E2 or S, is cleaved to S1 and S2 subunits and forms the large club-shaped peplomers on the surface of the virion which gives the virus its characteristic appearance. The El glycoprotein is believed to be responsible for determining the intracellular site of viral morphogenesis. A smaller group of coronaviruses contain a third membrane glycoprotein (Hogue and Brian, 1986, King et al., 1985, Pocock and Garwes, 1977), E3 or H, which, in the case of bovine Coronavirus , exhibits haemagglutinating and acetylesterase activities.