Comparative studies of piscine and amphibian iridoviruses.

A total of 30 iridoviruses collected from Australia, South-East Asia, North America, South America and Europe were characterised. With the exception of the South-East Asian iridoviruses all viruses were found to belong to the genus Ranavirus. All viruses, except those originating from South-East Asia, cross-reacted with antisera against epizootic haematopoietic necrosis virus (EHNV). Viruses or virus-infected cells were examined using electron microscopy, SDS PAGE, restriction endonuclease (RE) digestion, DNA hybridisation, and DNA sequencing. Data from RE digestion of genomic DNA, and from the sequencing of PCR products indicated that the viruses generally grouped according to their geographic and taxonomic (i.e. amphibian or fish) origin. The one exception to this was the viruses from the United Kingdom that grouped with the North American ranaviruses. The differences between specified genomic regions were small. To assess the validity of the differences in sequence homology, similar studies were performed with different isolates from two viruses (EHNV and Guatopo virus (GV), collected from different animals at different locations and time). The sequence data showed complete homology for the isolates for any one virus over the 200 and 586 bp regions examined. Collectively, the data showed that the coding region for the major coat protein (MCP) is stable for any one species (e.g. EHNV).

Further observations on the epidemiology and spread of epizootic haematopoietic necrosis virus (EHNV) in farmed rainbow trout Oncorhynchus mykiss in southeastern Australia and a recommended sampling strategy for surveillance.

Epizootic haematopoietic necrosis virus (EHNV) is an iridovirus confined to Australia and is known only from rainbow trout Oncorhynchus mykiss and redfin perch Perca fluviatilis. Outbreaks of disease caused by EHNV in trout populations have invariably been of low severity, affecting only 0+ post-hatchery phase fingerlings < 125 mm in length. To date the virus has been demonstrated in very few live in-contact fish, and anti-EHNV antibodies have not been found in survivors of outbreaks, suggesting low infectivity but high case fatality rates in trout. During an on-going study on an endemically infected farm (Farm A) in the Murrumbidgee River catchment of southeastern New South Wales, EHNV infection was demonstrated in 4 to 6 wk old trout fingerlings in the hatchery as well as in 1+ to 2+ grower fish. During a separate investigation of mortalities in 1+ to 2+ trout on Farm B in the Shoalhaven River catchment in southeastern New South Wales, EHNV infection was demonstrated in both fingerlings and adult fish in association with nocardiosis. A 0.7% prevalence of antibodies against EHNV was detected by ELISA in the serum of grower fish at this time, providing the first evidence that EHNV might not kill all infected trout. EHNV infection on Farm B occurred after transfer of fingerlings from Farm C in the Murrumbidgee river catchment. When investigated, there were no obvious signs of diseases on Farm C. 'Routine' mortalities were collected over 10 d on Farm C and EHNV was detected in 2.1% of 190 fish. Tracing investigations of sources of supply of fingerlings to Farm B also led to investigation of Farm D in Victoria, where the prevalence of anti-EHNV antibodies in 3+ to 4+ fish was 1.3%. The results of this study indicate that EHNV may be found in trout in all age classes, need not be associated with clinically detectable disease in the population, can be transferred with shipments of live fish, can be detected in a small proportion of 'routine' mortalities and may be associated with specific antibodies in a small proportion of older fish. Sampling to detect EHNV for certification purposes should be based on examination of 'routine' mortalities rather than random samples of live fish. Antigen-capture ELISA can be used as a cost effective screening test to detect EHNV on a farm provided that sampling rates conform with statistical principles.

Giant toads Bufo marinus in Australia and Venezuela have antibodies against 'ranaviruses'.

A serological survey was conducted for antibodies against 'ranaviruses' in the giant toad Bufo marinus in Australia and Venezuela. Sera containing antibodies against 'ranaviruses' were found in both countries. In Australia positive antibodies were identified in populations throughout most of the known range of B. marinus. Results were confirmed by immunofluorescence and immunoelectron microscopy where a characteristic staining pattern of 'ranaviruses' in infected cells was observed. Whilst a 'ranavirus(es)' has been isolated from populations of B. marinus in Venezuela, no virus has been isolated from Australian B. marinus populations. The significance of 'ranavirus' sero-positive B. marinus in Australia is discussed.

Isolation and characterization of iridoviruses from the giant toad Bufo marinus in Venezuela.

In this communication we describe for the first time the isolation of 7 iridoviruses from the toad Bufo marinus and an unknown species of frog Leptodactylus in Venezuela, South America. The viruses are icosahedral with electron-dense cores, each of which is surrounded by an inner membrane, capsid and a cell-derived envelope. The virus(es) have an average vertex to vertex diameter of 160 nm and replicate in the cytoplasm of a range of cell lines. Within the cytoplasm of infected cells, rarefied areas could be observed; structures lacked cellular organelles and contained complete, empty and developing viruses. Results from antigen-capture enzyme-linked immunosorbent assays (ELISA) with polyclonal antibody raised against epizootic haematopoietic necrosis virus (EHNV) indicated cross-reactivity between these isolates, Bohle iridovirus (BIV) and frog virus 3 (FV3). Comparison of polypeptide and genomic profiles indicated that the Venezuelan viruses shared many polypeptides of equivalent molecular weight with type species FV3. There were, however, differences between the group of Venezuelan viruses and FV3 and BIV. The viruses belongs to the family Iridoviridae and the genus Ranavirus.

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus).

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.

The effect of interleukin 1 alpha on acetaminophen-induced hepatotoxicity.

The protective effect of interleukin 1 alpha (IL-1 alpha) in mice with acetaminophen (AAP)-induced hepatitis was investigated. IL-1 alpha had a significant protective effect if given 2 or more hours (up to 24 hours) before AAP; it significantly reduced mortality of mice and decreased serum transaminase level. The maximal effect was obtained with the dose of 1000 U (166 ng/kg) IL-1 alpha. Pretreatment with IL-1 significantly increased the synthesis of prostaglandin E2 (PGE2) in samples of liver tissue from AAP-treated mice, but had no effect on the synthesis of leukotriene C4 (LTC4). Pretreatment with indomethacin (IMC) did not abrogate significantly the protective effect of IL-1. Thus, the hepatoprotective effect of IL-1 alpha can not be entirely explained by the stimulation of prostaglandin (PG) synthesis.

NK cell activity and estrogen hormone levels during normal human pregnancy.

NK cell activity was determined in peripheral blood of 24 women during pregnancy, and compared to NK activity of 40 healthy nonpregnant women in generative age. An increase in the first trimester was followed by a significant decline of NK activity in the second trimester, and a further fall in the third trimester of pregnancy. The initial rise of NK activity was predominantly due to primigravidas, whereas the fall in the second trimester was mainly due to multigravidas. There was a significant negative correlation between NK activity and the increasing levels of estrogen hormones (beta-estradiol, estriol and estrone) in the sera of pregnant women. However, when analyzed for each trimester of pregnancy separately, a significant (p less than 0.02) negative correlation was only found with beta-estradiol, suggesting that high doses of this hormone could contribute to pregnancy-associated NK suppression.