D. Berryman

Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses.

Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia. The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV). Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV. The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione. The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV. The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera. The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera. The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses. The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle.

Lack of Seed Coat Contamination with Cucumber mosaic virus in Lupin Permits Reliable, Large-Scale Detection of Seed Transmission in Seed Samples

Sowing seed stocks with minimal virus content provides a key control measure in preventing damaging epidemics of Cucumber mosaic virus (CMV) in crops of narrow-leafed lupin (Lupinus angustifolius). A seed testing service provides an estimate of percent CMV infection based on a dry seed test in which bulked subsamples of ungerminated seed are ground to a fine powder for testing. When enzyme-linked immunosorbent assay (ELISA) was used, CMV antiserum that gave low background optical density (A405) values with extracts of powder from subsamples of healthy seed provided greatest accuracy, readily detecting one infected seed in subsamples of 100 seeds. In comparative ELISAs on duplicate subsamples from eight different seed stocks, germination and dry seed tests always gave similar percent infection values. When seed coats were separated from the embryos of CMV-infected and healthy lupin seeds before testing by ELISA, the virus was only detected in embryos from infected seeds and never in their seed coats. Treatment with trisodium phosphate did not alter the low ELISA optical density (A405) values obtained with seed coats separated from infected seeds. Therefore, seed coat contamination with CMV is lacking in lupin, justifying large-scale routine use of a dry seed test to estimate percent virus infection in commercial seed samples.

Influence of glucocorticoids, neuregulin-1β, and sex on surfactant phospholipid secretion from type II cells

Glucocorticoids induce lung fibroblasts to produce fibroblast-pneumocyte factor, a peptide that stimulates type II cells to synthesize pulmonary surfactant. This effect is known to be more apparent in cells derived from female fetuses, a characteristic that has been attributed to sex-linked differences in the fibroblasts. In the current study, it has been shown that dexamethasone enhances both β-adrenergic receptor (β-AR) activity (1.3- to 1.6-fold increase) and (-)-isoproterenol-induced secretion of surfactant (1.8- to 1.9-fold increase) in type II cells. However, fibroblast-conditioned media (FCM), prepared in the presence of dexamethasone, generates a much greater response to (-)-isoproterenol (3.1- to 3.8-fold increase). Furthermore, each of these effects is more pronounced if both cell types are female-derived. It is hypothesized that the enhanced response to glucocorticoids is the result of a synergistic effect between the steroid and a component of FCM. Neuregulin-1β (NRG1β), which is elevated in FCM generated in the presence of dexamethasone, influences not only the rate of surfactant secretion and the β-AR activity in type II cells, but also enhances in both sexes the cellular response to (-)-isoproterenol. These results suggest that NRG1β might be more effective than glucocorticoids in treating prematurely born male infants, which are known to respond poorly to glucocorticoids. Given that glucocorticoids are known to induce higher levels of β-AR mRNA, the effect of NRG1β, alone and in combination with dexamethasone, on β-AR gene expression was measured using qRT-PCR. Whereas NRG1β had no effect alone, in combination with dexamethasone it produced up to a 4.2-fold elevation in the level of β-AR mRNA.

Role of neuregulin-1β in dexamethasone-enhanced surfactant synthesis in fetal type II cells

It is well established that glucocorticoids elevate the production of fibroblast‐pneumocyte factor (FPF), which induces type II cells to synthesize surfactant phospholipids. FPF, however, has not been identified and it is not clear whether it is a single factor or a complex mixture of factors. In this study it has been shown that, when lung fibroblasts are exposed to dexamethasone, the concentration of neuregulin‐1β (NRG1β) in conditioned medium is elevated 2‐fold (P < 0.05), even though NRG1β gene expression is unaffected. This, together with the finding that exposure of type II cells to NRG1β directly stimulates by 3‐fold the rate of phospholipid synthesis (P < 0.05), suggests that NRG1β is a component of FPF that promotes lung development.