A cytopathic viral agent was isolated from the pooled viscera of three moribund 0-f juvenile redfin perch (2-5cni-3'5cm), Perca fluviatilis L., collected from Lake Nillahcootie, near Benalla, Victoria, Australia. The fish were collected during investigations into the death of large numbers of juvenile redfin perch in artificial water impoundments in north-east Victoria during November and December 1984. Many fish displayed focal hepatocellular and haematopoietic necrosis.Pooled kidney, liver and spleen samples were homogenized with a chilled mortar and pestle, diluted 1:20 with Hanks' balanced salt solution containing penicillin and streptomycin, and centrifuged for 25 min at 3500g. Supernatant aliquots of 0-2 ml were inoculated into duplicate 5-cm^ drained confluent monolayers of RTG-2 (rainbow trout gonad) cells for 1 h at 25°C. The inoculum was then poured off and replaced with Eagle's minimum essential medium (pH7-4) containing Earle's Salts and 2% foetal calf serum, and the cultures incubated at 15'^C. A cytopathic effect (CPE) was first detected at 10 days post-inoculation and was reproduced by inoculation of supernatant fluid into fresh RTG-2 cultures. The CPE appeared progressively earlier with serial passages until by pass 10 the initial focal rounding of cells was detectable 24 h after inoculation. The CPE took the form of focal rounding followed by puncturing of the cell sheet and the development of plaques by progressive rounding, detachment and degeneration or lysis of the perimeter cells (Fig. 1). The agent was cytopathic for eight fish cell lines or primary cultures tested though the nature and development rate of the CPE, and titres reached, varied (Table 1). The highest titres were achieved using FHM (fathead minnow) cells, with a titre of 10^'T CID5oml attained in 7 days. Treatment of supernatant fluid containing lO' TCIDsoml of virus with 50% (v/v) ether for 60 min reduced the titre to 10^ TCIDsoml.Electron microscopy was performed on pellets produced by freeze-thawing and centrifugation of infected tissue-culture supernatants at 8000^ and 80000 g for 15 min and 90 min respectively. The pellets were resuspended in phosphate-buffered saline and prepared for electron microscopy by the pseudoreplica technique. Grids were negatively stained with 2% or 3% phospho-tungstic acid (PTA) at pH 6-0, 6-8 and 7 2. Particles ranging from 165 nm to 172 nm in diameter (distance between opposite vertices) were
. The epizootiology of epizootic haematopoietic necrosis virus (EHNV) was examined by experimental transmission and pathogenicity trials in the first known host species, Perca fluviatilis L., and 11 other introduced and native teleosts of Australia. Perca fluviatilis and four other species were highly susceptible to disease following bath exposure to EHNV. Three species developed disease only when inoculated by intraperitoneal injection, and four species wore resistant to infection under the experimental conditions employed. Renal haematopoietic necrosis was the most consistent lesion produced, with hepatic, splenic and pancreatic necrosis also occurring in certain species. The resistance of the virus to desiccation, disinfectants, high and low pH and temperature, and to prolonged storage at selected temperatures was assessed, demonstrating that the virus can survive for long periods in the aquatic environment and on fomites. Carrier individuals of P. fluviatilis, Salmo gairdneri Richardson, Maccullochella peeli (Mitchell), and possibly other teleosts, represent a further reservoir of infection. However, the virus could not be isolated from common insect and crustacean prey species, and no parasitic vectors were identified. It is proposed that EHNV has contributed to serious population declines of native species in recent decades, particularly of Macquaria australasica (Cuvier), Galaxias olidus Günther and Bidyanus bidyanus (Mitchell). Salmonids are also known to be naturally and experimentally susceptible to the virus.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.