Characteristics of a new reovirus isolated from epizootic ulcerative syndrome infected snakehead fish.

Epizootic ulcerative syndrome (EUS) has been infecting a wide range of fishes in the South and Southeast Asia for the last 2 decades. One reovirus-like agent (snakehead reovirus, SKRV), isolated from an EUS-infected snakehead fish and investigated in the present study, is the only reovirus so far isolated from an EUS-infected fish. SKRV was characterised by the presence of a double-stranded RNA genome with icosahedral symmetry and double capsid. The virus had an average size of 71 nm, a buoyant density of 1.36 g ml(-1) in CsCl and lacked a lipid-containing envelope. Apart from the above, the presence of a segmented genome and structural proteins falling into 3 specific size classes confirmed that the virus belongs to the family Reoviridae. SKRV differed from aquareoviruses by the lack of a cytopathic effect (CPE) with syncitium formation and in the segmentation pattern of RNA genome. The resistance to pH (3.0 to 9.0) and heat treatment and inability to multiply in mammalian cell lines and haemagglutinate human 'O' red blood cells (RBCs) differentiated SKRV from the rest of the similar genera in the family Reoviridae. Serological comparison indicated the antigenic distinctness of the isolate from selected American and European aquareoviruses. SKRV grew well in SSN-1 and SSN-3 cells at 25 to 30 degrees C but not in the most common Aquareovirus susceptible coldwater fish cell line--CHSE-214.

Cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, Dicentrarchus labrax.

A virus causing a vacuolating encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax, was isolated from brain tissue in a fish cell line (SSN-1) derived from striped snakehead, Channa striatus. The isometric, non-enveloped, 30 nm diameter virus particles were resistant to pH 2-9 and heating at 56 degrees C for 30 min. Infectious particles had a buoyant density of approximately 1.31 g/cm3 in CsCl. Two structural polypeptides of molecular mass 40 and 42 kDa were identified and the ssRNA consisted of two fragments of molecular mass 1.10 and 0.51 x 10(6) Da. From these characteristics the virus was identified as a nodavirus. Due to the broad range of susceptible fish hosts and the consistent neuropathology of the disease condition, the generic term piscine neuropathy nodavirus (PNN) is proposed for this infectious agent.

Complete nucleotide sequence and transcriptional analysis of snakehead fish retrovirus.

The complete genome of the snakehead fish retrovirus has been cloned and sequenced, and its transcriptional profile in cell culture has been determined. The 11.2-kb provirus displays a complex expression pattern capable of encoding accessory proteins and is unique in the predicted location of the env initiation codon and signal peptide upstream of gag and the common splice donor site. The virus is distinguishable from all known retrovirus groups by the presence of an arginine tRNA primer binding site. The coding regions are highly divergent and show a number of unusual characteristics, including a large Gag coiled-coil region, a Pol domain of unknown function, and a long, lentiviral-like, Env cytoplasmic domain. Phylogenetic analysis of the Pol sequence emphasizes the divergent nature of the virus from the avian and mammalian retroviruses. The snakehead virus is also distinct from a previously characterized complex fish retrovirus, suggesting that discrete groups of these viruses have yet to be identified in the lower vertebrates.

Viruses associated with the epizootic ulcerative syndrome (EUS) of fish in South-East Asia.

A number of birnaviruses, rhabdoviruses and a reovirus have been isolated from occasional fish affected with the epizootic ulcerative syndrome (EUS) in the past decade. The heterogeneous nature of these isolates, together with a low and inconsistent level of recovery from diseases specimens, suggests that these viruses may only represent adventitious infections unrelated to outbreaks of EUS. Furthermore, experimental induction of the condition by direct exposure to cell culture isolated viruses has not been achieved. The significance, if any, of C-type retroviruses identified in cell cultures derived from EUS-susceptible fish species is not known.

Spontaneously productive C-type retrovirus infection of fish cell lines.

The spontaneous production and release of morphologically typical, 85 to 90 nm diameter C-type retrovirus particles from four cell lines derived from three species of warmwater fish have been identified. Virus pellets from cell culture supernatants showed high levels of Mn(2+)-dependent reverse transcriptase activity at 24 degrees C. Peak enzyme activity was associated with a 1 x 16 g/ml sucrose gradient fraction. All four isolates induced a cytolytic infection of a bluegill fry cell line within 6 to 10 days.

Bacterial diseases of marine fish.

The principal bacterial diseases found among wild and cultured marine fish are reviewed. The bacterial agents discussed include the Gram-negative pathogens in the Vibrio, Aeromonas, Pasteurella and Edwardsiella genera, Renibacterium salmoninarum and the myxobacteria, streptococci, mycobacteria, nocardias and anaerobic organisms which have been associated with fish diseases. Methods for the isolation and identification of these organisms are described.

The fatty acid compositions of established fish cell lines after long-term culture in mammalian sera.

The effect of long-term culture of fish cells in mammalian serum on the phospholipid fatty acid composition was investigated. All the cell lines studied had much lower levels of polyunsaturated fatty acids (PUFA) than those found in intact fish tissues. In particular (n-3) PUFA were considerably depleted in the cultured cell lines, leading to very low (n-3)/(n-6) ratios in all the phospholipid classes. In general the cells were rich in 18:1, 16:0, 18:0 and 16:1 with 20:4(n-6) and 22:6(n-3) as the major PUFA. The fatty acid composition reflected the composition of the fetal calf serum added to the media rather than their fish tissue origins. The results were discussed in relation to the roles of PUFA in general cell metabolism and more specifically the role of (n-3) PUFA in fish cells.

Sterility testing of immunological products: comparative efficacy of media and effect of preservatives.

A comparison was made of the abilities of various culture media to support the growth of a range of micro-organisms commonly recommended as control strains in tests for the sterility of immunological products. The effects of phenol, cresol, formaldehyde and thiomersal on the growth of these organisms were studied. Attention is drawn to some limitations of the current pharmacopoeial test methods.

An inactivated, oil-emulsion vaccine for the prevention of porcine parvovirus-induced reproductive failure.

Pig fetuses inoculated at 45 days gestation with virulent porcine parvovirus (PPV) were harvested 10 days later. Virus was extracted, inactivated with binary ethylenimine and the antigen suspension emulsified with mineral oil adjuvant. One dose of this vaccine, or two doses with a 14 day interval, stimulated high and long lasting serum antibody titres in gilts. Vaccination caused no clinical reactions and lesions at injection sites were minor. Vaccination of seronegative gilts at 40 days gestation caused no adverse effects on fetuses. Six gilts which had been vaccinated five to nine weeks before mating were challenged intravenously with live, virulent PPV at 40 days gestation. At 98 days gestation 78 out of 84 (93 per cent) fetuses were alive and normal and no evidence of PPV infection was found in the six dead (mummified) fetuses. In four unvaccinated gilts similarly challenged with PPV at 40 days gestation only five out of 51 (10 per cent) fetuses survived to 98 days gestation and the virus was detected in 41 of the 46 dead (mummified) fetuses. This vaccine appears to be safe and effective for prevention of PPV-induced fetal loss in gilts.

Safety and efficacy of live and inactivated infectious bovine rhinotracheitis vaccines.

Two commercial live virus infectious bovine rhinotracheitis (IBR) vaccines for intranasal administration and an inactivated polyvalent calf pneumonia vaccine were compared for safety and efficacy in calves against experimental IBR infections. All three products were clinically safe for use in young calves; a mild, transient, febrile response was induced by one of the live vaccines. Vaccinal virus was recovered for up to 16 days after vaccination from nasal secretions of all calves given live vaccine. Both live vaccines stimulated a serum neutralising antibody response, but the inactivated vaccine failed to elicit any serological response. Following intranasal challenge four months after the first dose of vaccine, all live virus vaccinates remained systemically healthy. A slight nasal discharge and a few rapidly healing ulcers of the nasal mucosa were the only abnormalities observed. Both the group given the inactivated vaccine and the unvaccinated controls developed clinical IBR with pyrexia, ocular and nasal discharges, severe ulceration of the nasal mucosa and tracheitis and tachypnoea to varying degrees of severity. Parenteral administration of dexamethasone six months after challenge induced reactivation of virus shedding followed by a rise in humoral antibody titre irrespective of the original vaccination history.