At the request of Singapore netcage mariculture farmers of greasy grouper (Epinephelus tauvina) and seabass (Lates calcarifer) complaining of high mortalities in 2-4 cm juve niles, viral isolation from clinically affected fish was initiated. During 1986 to 1991 five viruses producing intracytoplasmic CPE were isolated from grouper and seabass in a seabass cell line (SB) which was immortalised from primary seabass larvae cell cultures". Electronmicroscopy (EM) revealed small (approximately 30 nm) icosahedral virions. Three grouper and one seabass viral isolates were obtained from juvenile fish (2-4 cm in length) which had abdominal distension and mani festations of swimming abnormalities. Only one isolate was from market-sized 31-32 cm groupers. All these five isolates were completely neutralised in the serum neutralisation test using a rabbit anti-striped jack nervous necrosis virus (SJNNV) serum. Further confirmation was made by use of the oligo nucleotide primer set designed for the amplification of T4 target region (426 bp) of SJNNV coat protein gene2) in a reverse transcription-polymerase chain reaction (RT-PCR). The PCR method effectively established the identity between our isolates and SJNNV.Because early attempts at isolation in BF-2 (ATCC CCL 91) were not successful, the SB was developed and by a process of passaging viral isolates can achieve titres of 10

Encephalitis caused by avirulent Semliki Forest virus was studied at regular intervals for 6 weeks in experimentally infected mice. The most obvious lesion was perivascular cuffing. Microglia were predominant near the vessels early in the infection. The cells of the cuffs were mostly lymphocytes. Although the virus produced acute encephalitis, which gradually resolved by 6 weeks, at no time were there abnormal neurological clinical signs. The cells of the brain and the immune response reacted to eliminate the virus.

Adult mice given two or three intraperitoneal inoculations with avirulent Semliki Forest Virus showed typical lesions of a viral encephalitis similar to those caused by a virulent strain of the virus. Demyelination also was seen in the medulla and in the folia of the cerebellar white matter. Neuronophagia was seen only in mice that had had three successive infections. The repeated inoculations of avirulent virus exacerbates the encephalitis of a single inoculation and causes demyelination. The mice did not have neurological clinical signs except for a short-lived weakness of the hind legs. No central nervous system lesions were seen by the 7th and 8th week after the initial infection and all mice recovered.

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An ultrastructural study of cerebellar lesions involving axonal and myelin degeneration induced in adult Swiss A2G mice by 3 intraperitoneal inoculations of avirulent Semliki Forest virus is described. Cerebellar white matter samples examined by light and electron miscroscopy 21 days after the first virus infection revealed microcystic areas, degenerating axons, macrophages containing myelin debris as well as normal elements. By light microscopy foci of myelin loss were apparent. At 28 days groups of degenerating axons were apparent in the white matter. No virus was seen in any of the sections.