Observations on the pathogenesis of bluetongue virus infections in sheep.

This study was instigated to provide basic information on the pathogenesis of bluetongue virus infection in sheep (and, where considered appropriate, to include cattle and goats) in support of other studies at the Animal Virus Research Institute, Pirbright, on the epidemiology and control of bluetongue infections. The investigation was conducted in four parts: 1) distribution of virus in infected sheep (a sequential slaughter study), 2) haematology of infected sheep, goats and cattle, 3) replication of virus in organ culture and 4) the growth of virus in cells derived from the haemopoietic system of sheep, goats and cattle. From the study on virus distribution in sheep it was evident that the lymphoreticular system was involved in the primary location and replication of virus. Once a viraemia was detected, virus was isolated from a large number of tissues and organs. The major haematological feature of bluetongue virus infection in sheep, goats and cattle was a transient pan-leucopenia; maximum leucopenia preceded peak viraemia and peak pyrexia. Studies on the type of the viraemia revealed, it to be cell associated with both the erythrocyte and leucocyte fraction. For technical reasons, attempts to ascertain the identity of the cell types involved in the leucocyte fraction were inconclusive since the techniques for cell separation were not sufficiently specific. This meant that although the preparations were relatively pure the number of infected cells in each cell population was below that of the contaminating cells; therefore it was not possible to say which of the cell populations were infected. Organ culture studies indicated that most tissues and organs were capable of supporting virus replication, in particular lymph nodes and other tissues and organs of the haemopoietic system with the exception of the liver. Prior to studies conducted on virus replication in various cell types (especially those isolated from the haemopoietic system) methods for the production, separation and purification of these cells were assessed. Where possible a basic technique was established for a particular cell type from any of the species of animal examined. Of importance during these preliminary experiments was the successful production of established macrophage cell lines derived from mononuclear cells from various sources:- alveolar, bone marrow, spleen, peripheral blood and mammary gland. These cell lines originated from sheep, goats and cattle. (See Appendix I). In studying the susceptibility of the various populations of cells to the virus it was found that endothelial cells from the vascular systems of sheep, goats, and cattle supported the growth of bluetongue at high and low multiplicities of infection and exhibited a cytopathic effect. Monocytes and macrophages from these three species of animal also supported growth of virus at high and low multiplicities of virus, and exhibited a cytopathic effect. An important difference was observed in macrophages of goat and cattle at low multiplicities; whilst these cells were susceptible there is evidence that they become persistently infected with low levels of virus being released without cytopathic effect occurring for at least 12 days post infection. The importance of this finding is discussed in relationship to overwintering mechanisms for bluetongue in goats and cattle in certain parts of the world. The following conclusions were made from the study in this thesis. 1. Bluetongue replicates in the haemopoietic system namely a) primary replication occurs in the lymph nodes and other tissues of the lymphoreticular system. b) the cells involved in the primary replication are probably the avidly phagocytic cells of the mononuclear phagocytic system which includes monocytes and macrophages. c) endothelial cells of blood vessels are involved in replication of the virus and d) neutrophils may be involved in virus replication. 2. Monocytes and macrophages are probably involved in the mechanism of persistent infections in cattle and goats. On the basis of this study areas of future studies are discussed and these fall into three categories: 1) mechanisms of clinical disease 2) mechanisms of persistent infection and 3) vaccination