Development of gene probes to P virus (Reoviridae) for disease diagnosis in crustaceans

This study reports the development of two important techniques, gene probes and haemocyte cultures that have not been previously available to investigate viral diseases in temperate water marine decapods. These techniques were used to investigate numerous aspects of a reovirus infection of the swimming crab Liocarcinus depurator, P virus, both in vivo and in vitro. The construction and subsequent use of a gene probe has revealed that, not only can virus be experimentally transmitted to L. depurator by injection, but that it is present in natural populations of crabs from the North Sea. Seasonal variation in both incidence of P infection and in incubation time was observed. The incidence of infection increased with increasing temperature whereas incubation time decreased with increasing temperature. In vivo, P virus was found to cause marked haemocytopenia in infected L. depurator and a cytopathic effect, vacuolisation of haemocytes was observed. This effect was not observed in the haemocytes of the shore crab, Carcinus maenas, providing evidence that P virus does not infect this species. To address the lack of techniques for in vitro studies, a cell culture system for crustacean haemocytes was developed. Primary culture of two haemocyte types, hyaline and semi-granular haemocytes was established for haemocytes of both L. depurator and C. maenas. High haemocyte viability was obtained for at least two weeks and, cells retained their functional capabilities in vitro. Having successfully established a haemocyte culture system and the gene probe E2b, it was then possible to begin investigations on P virus infections in vitro. P virus produced a number of effects on haemocytes of L. depurator in vitro. Haemocyte number and haemocyte viability decreased after addition of P virus and a number of cytopathic effects were observed such as necrosis, pycnosis and vacuolisation