A. D. Hyatt

Lesions of Experimental Equine Morbillivirus Pneumonia in Horses

Laboratory examinations of equine morbillivirus included experimental reproductions of the disease caused by the virus by transmission of mixed lung and spleen taken from two field equine cases into two horses and by inoculating tissue culture virus into a further two horses. The most distinctive gross lesions of the diseases that developed in three of the horses was that of pulmonary edema characterized by gelatinous distension of subpleural lymphatics. Histologically, the lesions in the lungs were those of serofibrinous alveolar edema, alveolar macrophages, hemorrhage, thrombosis of capillaries, and syncytial cells. Clearly defined vascular lesions in three horses that became clinically affected within 8 days of inoculation of virus included intramural hemorrhage, edema, and necrosis and syncytial cells in the endothelium of pulmonary vessels (∼40-70 μm in diameter). Vascular lesions accompanied by parenchymal degeneration were also seen in the heart, kidney, brain, spleen, lymph node, and stomach. A fourth horse, which survived for 12 days, had detectable lesions only in the lungs, which were more chronic than those in the other three horses, a greater degree of cellular infiltration, and fewer well-defined vascular lesions. Sections stained by an indirect immunocytochemical method showed equine morbillivirus antigen was present in the vascular lesions and along alveolar walls. When endothelial cells were examined by electron microscope, cytoplasmic virus inclusion bodies containing filamentous structures were seen that reacted to an immunogold test to equine morbillivirus antigen. The presence of the syncytia in the small blood vessels in the lungs and other organs was interpreted as an important characteristic of the disease and consistent with a reaction to a morbillivirus.

The grid-cell-culture technique: the direct examination of virus-infected cells and progeny viruses.

We describe a method for the structural analysis and identification of viruses, without purification or concentration steps which could alter virus morphology. Virus‐infected cells grown on carbon‐Parlodion‐coated electron microscope grids release large numbers of progeny viruses which adsorb to the surface of the grid and are revealed by negative staining. The technique is rapid, sensitive and can be used at three levels. (1) Negative staining of whole cell preparations revealed both extracellular and intracellular viruses or nucleocapsids beneath the plasma membrane; (2) non‐ionic detergent extraction of cells infected with certain viruses reveals cytoskeleton‐associated, virus‐specific structures normally only observed after thin sectioning; (3) cultures prepared by either procedure are suitable for colloidal gold immunological studies. Extracellular and cytoskeletal‐associated viruses were heavily and specifically labelled with gold. The results indicate that the technique may be used to rapidly identify unknown viruses on the basis of size, topography, morphology and mode of maturation from the infected cell, as well as the presence of characteristic intracellular cytoskeletal‐associated structures. The technique also has potential use in the sero‐grouping and sero‐typing of viruses with specific monoclonal antibodies.

Epidemic of blindness in kangaroos--evidence of a viral aetiology.

Objective To determine the cause of an epidemic of blindness in kangaroos. Design and procedures Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. Sample population For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. Results Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. Conclusion Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.