MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15 Research and Development Final Project Report (Not to be used for LINK projects) Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit MAFF, Area 6/01 1A Page Street, London SW1P 4PQ An electronic version should be e-mailed to [email protected] Project title Research on viruses of fish and shellfish MAFF project code FC1105 Contractor organisation CEFAS Weymouth Laboratory and location Barrack Road The Nothe Weymouth Dorset DT4 8UB Total MAFF project costs £ Project start date 01/04/96 Project end date 31/03/00 Executive summary (maximum 2 sides A4) Viruses cause many serious diseases in both wild and cultivated fish in the freshwater and marine environments. The diseases are of importance because they are can cause very high mortality in fish populations, can spread rapidly and cannot be controlled by therapeutic measures. In Great Britain they are controlled by movement restrictions and by control of importation of fish and fish products, and to that end several virus diseases have been made notifiable: viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious salmon anaemia (ISA), spring viraemia of carp (SVC) and infectious pancreatic necrosis (IPN) in salmon. The aims of this project were to support the policy objectives of preventing the introduction and spread of serious fish and shellfish diseases in Great Britain, improving methods of dealing with diseases, and helping to create the conditions in which an efficient GB fish/shellfish industry can develop and in which freshwater fisheries can flourish. This was achieved by characterisation of newly isolated strains/variants of viruses, monitoring and improving methods for detection and identification of the viruses, and monitoring non-notifiable virus diseases exotic to GB to determine the threat they pose to GB fish stocks. Much of the work concerned virus diseases of cyprinids, and one of the problems that was resolved was the relationship between the virus causing the notifiable disease SVC (SVCV) and the non-notifiable pike fry rhabdovirus (PFR). The two viruses are reported to have overlapping host ranges: SVCV affects carp and other species, mainly cyprinids, and according to the scientific literature, PFR affects pike, cyprinids (but not carp under natural conditions) and other fish species. During investigations into cyprinid mortalities, viruses have been isolated at the laboratory that, on the basis of serological tests, were related to both SVCV and PFR but could not be classified as either; they were provisionally termed “intermediate” viruses. Because they were associated with cyprinid mortalities it was important to determine their relationship to SVCV, so as to CSG 15 (Rev. 12/99) 1 Project Research on viruses of fish and shellfish MAFF FC1105 title project code provide advice as to whether they should be controlled. The isolates were compared by immunoassays and neutralisation tests using antisera against SVCV and PFR, and by nucleotide sequence analysis. The isolates could be allocated to one of three related groups by the serological tests, but the precise grouping of certain isolates varied according to which serological test was used. However, nucleotide sequence comparisons showed that they formed four distinct genogroups. The majority of the viruses were isolates of SVCV, which formed Genogroup I. The bulk of the other isolates, from fish such as tench, roach and bream, formed Genogroup IV. Only two isolates were classified as PFR (Genogroup III), and a single isolate from grass carp formed Genogroup II. Many isolates obtained from other laboratories, and originally classified as PFR, were allocated to Genogroup IV. Although the isolates in Genogroups II, III and IV are distinct from SVCV, many have been associated with SVC-like diseases and high mortality, particularly the isolates in Genogroup IV. We are recommending to the scientific community that the name for the disease caused by all these isolates should be spring viraemia of cyprinids, and that spring viraemia of carp is no longer an appropriate name. An immunoassay for detecting SVCV antibodies in cyprinids that had been developed prior to this project has been used to screen several hundred carp sera. Antibodies were found but they could not be correlated with the presence of viruses in antibody-positive fish. This does not mean that the immunoassay is prone to giving false positive results, but taken together with preliminary results from in situ hybridisation studies may mean that virus isolation in cell culture is a less sensitive method, or is targeting the wrong tissues when testing for carrier fish. As virus isolation in cell culture is the currently accepted method for detecting SVCV-positive fish, that work should be continued. Immunoassays for detecting SVCV directly in infected fish tissues were refined and can be used as preliminary diagnostic tests in suspected outbreaks of those diseases. This means that control measures to stop the movement of infected fish can be applied sooner and thus help reduce the spread of the disease. Work has been done to identify the cause of mortalities reported in koi carp, but no viruses were isolated in diagnostic tests. However in the USA, a herpesvirus was isolated from similarly affected koi carp, and that virus was obtained for familiarisation studies. Work with that virus has been impeded because it has only been cultured in one specific cell line derived from koi carp. That cell line was obtained from it’s originators in the USA, and another koi carp cell line was developed at this laboratory; the latter cell line was also found to be susceptible to the herpesvirus. As this was only achieved toward the end of the project, further work will have to be continued in a new project. An agent has been isolated on many occasions during diagnostic testing on carp, and attempts were made to identify it. The agent is very slow growing and does not appear to be produced in large amounts. The agent has not yet been identified, and may not be a virus. It has usually been isolated from carp that have shown a wide and variable range of clinical and histpathological signs and may not be a primary pathogen, but only occur in them when they are stressed or suffering from other diseases. In recent years VHSV has been isolated from an increasing number of fish from the marine environment, including salmonids, but little is known about the properties of the viruses, and to what extent they differ form isolates from freshwater fish; it is known that the marine isolates are generally avirulent or of low virulence for salmonids, but can be virulent for marine fish. Two months prior to the start of this project VHSV was isolated at this laboratory from Atlantic herring from Rye Bay, and work commenced under this proposal to characterise that and other marine VHSV isolates. As VHSV had previously been considered a pathogen of freshwater fish, there had been no measurement of its survival time in the seawater, but that information is crucial when undertaking risk analyses of its spread in the marine environment. Laboratory tests were therefore undertaken to measure the survival time of nine VHSV isolates in seawater at 4, 10, 15 and 20°C. The survival time was inversely proportion to temperature, and ranged from 3 - 35 days at 4°C and from <1 – 7 days at 20°C for the different isolates. The preferred cell lines for the growth of the marine VHSV isolates was investigated, as there was evidence that one cell line used for isolating VHSV from freshwater fish was initially refractory to a VHSV isolate from a marine fish. The relative sensitivity of 12 fish cell lines to 13 VHSV isolates from both the marine and freshwater environments were compared. There were differences in susceptibility of the cell lines, but all four of the lines specified by the EU for screening for VHSV and IHNV (bluegill fry, BF-2, Epithelioma papulosum carpio, EPC, fathead minnow, FHM, and rainbow trout gonad, RTG-2) were susceptible to the isolates. The BF-2 and FHM cells were the most sensitive cell lines to VHSV isolates of any origin, whereas some marine isolates grew poorly in RTG-2 or EPC cells. Experiments were done to determine whether a laboratory test could be devised that might be used to predict whether a VHSV isolate was virulent or not. Preliminary tests showed that measuring the pH optimum for causing fusion in cell cultures after infection with a VHSV isolate may be of value, but work is needed with more isolates to confirm this. CSG 15 (1/00) 2 Project Research on viruses of fish and shellfish MAFF FC1105 title project code In 1997 and 1998 marine fish from around the coast of England and Wales were again screened for the presence of VHSV as part of a wider fish disease survey. VHSV was not isolated, but birnaviruses were isolated from Atlantic herring from Liverpool Bay and from Atlantic cod from Camarthen Bay and Rye Bay, and an aquareovirus was isolated from haddock from Off Amble. The fish from which the viruses were isolated did not exhibit any clinical signs of disease, and the significance of the viruses is not known. A watching brief has kept on the emergence and/or spread of virus diseases that may pose a threat to GB fish stocks. The salmon head kidney (SHK-1) cell line used for isolation of ISA virus and the striped snakehead (SSN-1) cell line used for isolation of nervous necrosis viruses have been obtained and used for familiarisation studies with the viruses and as diagnostic tools. This also enabled sensitivity and specificity testing of primers developed for the reverse transcription-polymerase chain reaction for those viruses under another project to be conducted.