Publications

Embryonated eggs were coinfected with two strains of the coronavirus avian infectious bronchitis virus (IBV), IBV-Beaudette and IBV-M41, to investigate whether recombination between the two strains would occur. Virions were isolated from the allantoic fluid of the coinfected eggs and putative hybrid RNAs were detected by polymerase chain reaction (PCR), using strain-specific oligonucleotides. PCR products, of the expected sizes, were obtained as predicted from potential recombination events between the nucleoprotein (N) gene and the 3'-untranslated region of the two IBV genomes. Sequencing confirmed that they corresponded to hybrid RNAs. Virus produced as a result of the mixed infection was treated with an M41-specific neutralizing monoclonal antibody and passaged in Vero cells, in which IBV-Beaudette, but not IBV-M41, replicated. Hybrid RNA was still detectable after three serial passages. Since no IBV-M41 was detectable this confirmed that infectious recombinant genomes had been produced in the embryonated eggs. These findings not only support the circumstantial evidence, from sequencing studies of IBV field strains, that recombination occurs during replication of IBV and contributes to the diversity of IBV, but also show that coronavirus RNA recombination is not limited to mouse hepatitis virus.

Nitric oxide (NO) was produced when bovine peripheral blood mononuclear cells (PBMC) or purified, adherent PBMC (macrophages) were incubated in vitro with bovine recombinant interferon gamma (Bo rIFN-?). NO was produced by cells from naive, uninfected calves as well as by cells from cattle either infected with or recovered from infection with Theileria annulata or Theileria parva. PBMC of cattle undergoing tropical theileriosis (T. annulata infection) or East Coast fever (T. parva infection) synthesized NO spontaneously in vitro. NO was also induced when PBMC of immune, but not of naive, cattle were cultured with T. annulata macroschizont-infected cell lines. Macrophages alone were not stimulated to produce NO by such infected cells. In vitro establishment of macroschizont-infected cell lines was suppressed either by incubating sporozoites with S-nitroso-N-acetyl-DL-penicillamine (SNAP), a NO releasing molecule, prior to invasion of PBMC or by pulsing developing cultures of trophozoite-infected cells with SNAP. Proliferation of established macroschizont-infected cell lines was not affected by SNAP. Taken together with the well documented roles of NO in neurotransmission, vasodilatation, cell and tissue damage and immunosuppression, the results presented here indicate that NO may not only protect cattle against T. annulata and T. parva but, if produced in excess, play a prominent role in the pathogenesis of tropical theileriosis and East Coast fever.

A 7-kb fragment of Streptomyces rochei A2 chromosomal DNA was cloned into pAT153 and shown to confer endoglucanase (Eg1S) activity on Escherichia coli cells. In E. coli clones, the Eg1S was secreted into the periplasm. Deletion analysis revealed that an 827-bp fragment was enough for the enzymatic activity. Sequence analysis showed that the 827-bp fragment codes for the catalytic domain of the enzyme. The complete sequence of the gene (eg1S) is 1149-bp long. A signal peptide, a catalytic domain and a cellulose-binding domain were identified from the nucleotide sequence, and the Eg1S found to belong to the family H of cellulase catalytic domains. These conclusions were substantiated by determination of the N-terminal sequence of the purified protein and zymogram analysis, which revealed protein species with a molecular mass equal to that deduced from the nt sequence analysis.