Cell-culture isolation techniques
Fixed rabies viruses can grow in a wide variety of cells. Successful in vitro cultivation of rabies virus was first reported in 1936. This property has been used extensively in research on rabies. However, it is only recently that techniques for the isolation of street rabies from suspect material in cell cultures have been developed. Tests for the isolation of street rabies in cell culture were first carried out in the mid 1970s using baby hamster kidney cells, line 21 (BHK-21), and chick embryo-related (CER) and neuroblastoma cells. These studies demonstrated that rabies infection could be detected by immunofluorescence from as early as 4-5 hours up to 5 days following inoculation. Furthermore, it was found that BHK-21 cells were comparable in sensitivity to mice, whereas neuroblastoma cells were more sensitive than mice to infection by street rabies virus. The difference in sensitivity between neuroblastoma cells and BHK-21 cells and other cell lines was reported to be associated with the neural origin of the former. However, Webster & Casey suggested that the difference may also be related to viral strain differences, as well as to cell type.
In routine rabies diagnosis, positive specimens contain amounts of antigen that can easily be detected by the FA test, the MI test or virus isolation in neuroblastoma cells. In the latter case, the result is obtained within 18-24 hours, although rabies antigen may be detected in these cells by FA as early as 4-5 hours after inoculation. Furthermore, virus isolation in cell culture has been shown to be as efficient as the FA test and the MI test for demonstrating small amounts of rabies virus. However, specimens containing a small amount of rabies virus and which are negative by FA and subsequently positive by virus isolation in cell culture require an incubation period of 4 days after inoculation of the cells.
In view of the usually short delay in obtaining the result, isolation of rabies virus in cell culture should replace intracerebral mouse inoculation whenever possible. It should, however, be borne in mind that only laboratories where cell-culture techniques are currently used can successfully maintain neuroblastoma cells for diagnosis.