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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

A SID 5A form must be completed where a project is paid on a monthly basis or against quarterly invoices. No SID 5A is required where payments are made at milestone points. When a SID 5A is required, no SID 5 form will be accepted without the accompanying SID 5A.

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ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code F1136

2. Project title

Research on the identification, diagnosis and significance of notifiable and emerging virus diseases of fish and shellfish

3. Contractororganisation(s)

     CEFAS Weymouth LaboratoryBarrack RoadThe Nothe Weymouth, DorsetDT4 8UB

54. Total Defra project costs £ 1366184

5. Project: start date................ 01 April 2000

end date................. 31 March 2005

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.This contract had a wide remit, which enabled us to respond quickly to new virus disease problems, and to changing Defra customer priorities. As the proposal was commencing Defra requested that we conduct a survey to determine whether ISAV could be detected in England and Wales, to complement a similar survey being conducted in Scotland. That survey had not been in the original proposal, and so other priorities/targets had to be adjusted accordingly. The survey, using virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) did not detect any ISAV-positive fish in England and Wales. Likewise in 2003-04, targets had to be revised to allow us to commence work on determining the sensitivity of a range of viruses to heat, acid and alkali. That was a preliminary to conducting field trials on the effectiveness of ensiling and composting methods to inactivate viruses contained in fish by-products. The work initially involved a calibration study with our counterparts in FRS Aberdeen with whom we collaborated in this work. The calibration was successfully achieved, and work here commenced on testing the susceptibility of 8 viruses to a temperature of 60°C and pH 4 and pH 12; The FRS laboratory tested another 8 viruses. The results from the two laboratories were amalgamated and the most resistant viruses were determined which were taken forward into field trials (see contract F1157 Final Report). The most resistant virus to 60°C was IPNV Sp, and to pH 12 was ISAV. The laboratory study showed that pH 4 was ineffective at inactivating several viruses, and field tests were not conducted using that pH.

Progress made against original objectives:

a) We have been successful in developing and validating new diagnostic methods. As part of a now-completed EU contract we validated a PCR and in situ hybridisation (ISH) method for oyster herpesvirus, and detected the virus in the UK in oyster samples collected in 2000, but not in 1999 or 2001. The ISH method has been developed for VHSV, SVCV (and similar vesiculoviruses) and KHV. It has yielded important information, particularly for the analysis of archive histological material. Development of an archive PCR complemented these studies. Work on the IHNV ISH was deferred to b) Five of six sites positive for the bream rhabdovirus in 1999 were re-sampled in 2000, but no virus was isolated. Two fish from one site were positive by RT-PCR, and 16/30 fish from that site were positive by ISH. Experimentally the virus was pathogenic for other cyprinids, particularly dace.

c) Measures were put in place to allow us to isolate and identify KHV, which include both isolation in cell culture and PCR. Cell culture isolation is less sensitive than PCR, and a nested PCR was developed to increase further the sensitivity of detection. Although the virus has been detected most frequently in koi carp, the virus was isolated from common carp during a disease epizootic in a fishery in 2003 and again from diseased carp from four fisheries in 2004. That is a serious development as it means that our feral carp populations are at risk from the disease. ISH and archive PCR studies suggested that

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the virus has been present in the UK since at least 1996, which was four years before we were able to demonstrate the presence of the virus in the UK by cell culture isolation. Work is ongoing, partially funded by the EU under the AVINSI contract, to identify antiviral genes in carp following infection with KHV. Libraries of potential genes have been created, and are currently being screened.

In recent years, strains of SVCV have been identified at the laboratory that differ in certain propereties from previous isolates. The majority of the newer strains were of Asian origin, but the recent SVCV isolates from the USA also have similar properties and are genetically similar to the Asian isolates. The Asian isolates were usually isolated during screening procedures, and only two UK isolates were associated with disease. Commercial test kits for the identification of SVCV were assessed. One kit based on the ELISA produced a number of false positives by reacting with non SVCV rhabdoviruses. The second kit, based on immunofluorescence using a monoclonal antibody gave many false negative results, by only reacting with a limited number of SVCV isolates tested.

Other work included investigations into spring carp mortality syndrome, identification of a virus frequently isolated from carp and investigations into an iridovirus isolated from dace.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

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The original scientific objectives were:

1. To validate new diagnostic methods (e.g. PCR, gene probes) for viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), cyprinid rhabdoviruses and oyster herpesvirus and to develop diagnostic procedures for emerging pathogens as appropriate.

2 To determine the prevalence of the bream rhabdovirus in selected sites in England and Wales.

3 To assess the impact of emerging pathogens (isolated in GB or abroad) on native fish or aquaculture species. Also to investigate novel viruses isolated during statutory disease monitoring (under FA001) and advise on their potential significance.

However, two new objectives were added at the request of the Defra customer:

4. To undertake a survey for infectious salmon anaemia virus (ISAV) in England and Wales in 2000-2001, and

5. To investigate the efficacy of composting and ensiling fish waste with regard to the inactivation of fish pathogenic bacteria and viruses in order to comply with the animal by-products regulations.

The two new targets were incorporated into the contract, and rather than drop existing objectives, all were kept in the proposal, but the targets were either delayed, or reduced in scope. The survey for ISAV was made the priority for the first year of the proposal, and so from the start, the planned targets and milestones of the proposal were changed.

At the end of Year 3 the targets were revised as follows, although they were provisional as the full impact of the work under new Objective 5 had not been fully determined:

Current milestone 02 / 04: Results of studies on emerging virus diseases. This will be primarily on KHV, and include work under the EU AVINSI project. Different populations of carp will be studied, and diagnostic methodology will be refined. Studies of archive material will continue.

Current milestone 03 / 04: Advice on significance of novel viruses isolated during disease monitoring. This will include characterisation of Asian SVCV isolates, particularly with respect to the ability of commercially available reagents to detect these isolates. Work will also include ensuring we have the diagnostic capability for sleeping disease/pancreas disease, that we can identify a putative picornavirus in molluscs, and further work to identify an unknown agent frequently isolated from cyprinids in diagnostic tests.

At the end of Year 4 the milestones were changed as follows:

Current milestone 01 / 05: Results of studies on emerging virus diseases. This will be primarily on KHV, and will include work under the EU AVINSI project. Virus-induced genes will be identified and cloned, and inhibitor of apoptosis genes will be identified. During the creation of gene libraries, further KHV genes may be identified. Diagnostic methodology will be refined and studies of archive material will continue. It is hoped that studies on KHV latency will be undertaken.

Current milestone 02 / 05: Advice on significance of novel viruses isolated during disease monitoring. This will include characterisation of SVCV isolates from different sub-genogroups. Work will also include improving our ability to detect a putative picornavirus in molluscs, completion of work to identify a possible myxovirus isolated from cyprinids in diagnostic tests, and further work on a possible new herpes-like virus from cyprinids.

Those milestones will be supplemented as follows:

New milestone 03 / 05: Determine the susceptibility of viral pathogens to heat and high and low pH in vitro, in conjunction with the FRS Laboratory, Aberdeen. Select the most resistant virus and undertake field trials of an ensiling system, and if available, a high pH treatment system.

New milestone 04 / 05: Results of further tests on susceptibility of wild fish to notifiable viruses. This will involve continuation of development and validation of detection methods such as in situ hybridisation and immunohistochemistry.

Methods and Results

New Objective 4. Survey for ISAV.

At the request of the MAFF (former name for Defra) customer a survey for the presence of infectious salmon anaemia virus (ISAV) was conducted in selected rivers in England and Wales in 2000-2001. The survey was conducted using methods agreed with the FRS Laboratory, Aberdeen which conducted a similar survey in Scotland over the same time period. In order to provide complete comparability between the two laboratories we obtained a salmon cell line (TO cells) from Norway which the FRS laboratory wanted to use as it had found them more sensitive than the SHK-1 cell line that this laboratory normally used, and we spent a short time building up stocks of the cells and familiarising ourselves with them. Prior to the survey, a calibration exercise was done to ensure that the results from the two laboratories would be comparable. Tissues from salmon which had died from ISA during experimental challenge were prepared in replicate by FRS, and ISAV-free tissues were also prepared. This was done by staff not directly concerned with ISA diagnostics in order to avoid bias. Replicate samples were sent to Weymouth and to the ISA diagnosticians at FRS under the same conditions of carriage. The samples were screened by ISAV reverse transcription polymerase chain reaction (RT-PCR) and ISAV virology, and complete agreement between the two laboratories was obtained.

Adult salmon were caught by commercial netsmen or were provided following holding in fresh water and stripping at hatcheries. Salmon parr were the target fish for testing in riverine habitats but where sufficient salmon were unavailable, brown trout were also sampled. These fish were obtained by electrofishing. Sea trout were fished from the estuaries of rivers, caught in the sea by gill netting and also obtained from commercial fisheries, hatcheries and traps. Fish were examined for gross clinical signs of ISA. The aim was to catch and test 30 fish at each location. Samples of kidney, liver, spleen and heart were taken from fish and transported from the sampling site to the laboratory in chilled transport medium. Fish were mostly sampled as pools of five fish but some fish were tested individually. A total of 751 Atlantic salmon and 276 brown trout/sea trout from 29 freshwater and 12 estuarine/seawater sites on 37 river systems (Table 1) were tested for ISAV in pools of up to five fish by isolation in cell culture and individually by the (RT-PCR). RNA was extracted using TriZol and ISAV was detected in the samples using two different primer sets. One was as described by Mjaaland et al. (1997), and the other was an in-house primer set designed by Dr David Stone used for comparison.

Table 1– Rivers sampled in England and Wales for the ISAV wild fish survey. The sites reflected locations where Atlantic salmon and brown trout / sea trout were likely to be obtained.

South West South Wales North Wales North West North East

1. Itchen2. Avon Water3. Avon

(Hants)4. Piddle5. Lynher6. Fowey7. Camel8. Lyn9. Dart 10. Exe 11. Test 12. Bray

1. Usk2. Tawe3. Loughor4. Tywi5. Taf6. W Cleddau7. Tiefi8. Rheidol9. Ogmore10. Clywedog

(Severn)11. Culn (Ely)

1. Llyfni2. Seiont3. Dee4. Mawddach

1. Kent2. Derwent3. Eden4. Esk5. Lune 6. Caldew

1. Tyne2. Coquet3. Tees4. Kielder

(Tyne)

No virus was isolated, and no fish produced an ISAV-specific PCR product. This work was labour intensive and relied heavily on input from a small number of individuals from the Weymouth Laboratory’s Fish Health Inspectorate who obtained the samples, and also input from staff in the Virology and Molecular Biology Functions at the Laboratory. Without their invaluable help the staff directly employed on this contract would not have been able to complete the survey in the time-scale required, and their assistance is gratefully acknowledged. This work was not in the original proposal to MAFF, but was undertaken for MAFF as a priority. That meant that other targets were not met, or were delayed, notably part of 01/01, part of 02/01, all of 03/01.

Objective 1. New Diagnostic Methods.

PCR for marine viral haemorrhagic septicaemia virus. It was intended that marine fish, particularly herring, would be collected from sites in Liverpool Bay in June 2000 and tested for viral haemorrhagic septicaemia virus (VHSV) by isolation in cell culture and PCR as part of a programme to validate the PCR, particularly with respect to virus isolates of marine origin. This target was delayed because of the survey for ISAV. However, in March 2001 the opportunity arose for marine fish to be collected for this work from four sites in the North Sea, and four sites in the German Bight. The samples were taken as part of an international collaborative programme Biological Effects of Contaminants in Pelagic Ecosystems (BECPELAG). The aim of BECPELAG was to apply a suite of methods to samples of fish and shellfish from from three sites in the vicinity of an oil platform and one reference site in the Statfjord area (near the Norwegian coast) , and three sites and a reference area on a contamination gradient in the German Bight (Figure 1) in order to try to determine whether there were any differences in pathologies, differences in immunological parameters, differences in cellular and sub-cellular biomarkers etc between the sampling areas. Some of the fish and shellfish had been placed in cages at the sites and others were free-living and were caught by trawling. Our part in the project was to determine whether VHSV could be isolated from caged juvenile herring from the different sites, and if successful, determine whether there was any correlation between virus prevalence and sampling area. As the programme developed it was apparent that there would not be sufficient caged juvenile herring for all the participants to test, and we were asked to test wild-caught herring from the different sites. We also included testing for ISAV as one of the sampling sites was close to Norway, where ISA occurs, and it has been suggested that herring may be a reservoir for the virus . Although herring were supposed to be abundant in all sampling areas, it was not possible to collect 30 herring from each sampling area as had been planned. Other fish species were collected instead. The fish sampled are listed in Table 2.

All 322 samples of marine fish (predominantly herring and whiting) were tested for the presence of VHSV and ISAV by isolation in cell culture, and for VHSV by RT-PCR. The viruses were not isolated from any sample, but a birnavirus was isolated from one whiting (a new host record for that virus). The samples were negative for VHSV by RT-PCR.

Oyster herpesvirus. We were partners in a European Union (EU) funded project (VINO, FAIR CT98-4334) to validate diagnostic tools for oyster herpesvirus (OsHV). Our role was to be the negative control for the the project as OsHV had not been identified in England and Wales, but had been identified in the countries of the other partners (France, Eire and Spain). Oysters and clams were collected from five sites in England and Wales from January to March 2000, August to October 2000, January to April 2001 and August –September 2001. These were in addition to samples collected in 1999 during a previous MAFF contract. Diseased animals were taken when available, but the majority of the animals were apparently healthy. A sample from each animal was analysed by the PCR for oyster herpesvirus (OsHV). Samples from Crassostrea gigas spat collected in January 2000 from one site and from Ostrea edulis larvae collected in March 2000 from a second site were positive by PCR. The nucleotide sequence of the PCR product of the samples confirmed them to be OsHV-1, having 94.4 % and 98.3 % homology, respectively, to reference OsHV DNA. All other samples were found to be negative for OsHV DNA.The samples were negative by histology, and in situ hybridisation (ISH) results were inconclusive. The summer/autumn 2000, and all the 2001 samples collected from every site, including the previously positive sites, were negative by the PCR; all the 1999 samples were negative.

Archive material of oysters collected from north Wales in 1976 following an episode of high mortality was re-examined, and the histological appearance was typical of OsHV infection. There was only a small amount of archive material available, which limited the work that could be done, with it, but one tissue section was analysed by ISH, and showed a positive reaction in a low number of cells. In March 2001 mortalities in oysters and clams from a hatchery in Guernsey were investigated. As part of that investigation samples fixed by hatchery personnel in 1997 and 2000 were also examined. Oysters fixed for electron microscopy in 1997 were positive for OsHV; samples taken in 2001 and tested by the PCR were negative for the virus. Examination by light microscopy revealed the presence of Dermocystidium-like organisms in samples of oysters taken in 1997 and 2000, and in clams sampled in 2001. The results show that OsHV, or a related virus, has occurred in certain UK stocks over a period of nearly 30 years, although we do not know whether the virus has been present continuously over that time. The virus is associated with high mortality, particularly in France, but no obnormal mortalities were reported at the sites where it was identified in England and Wales in 2000. Currently OsHV appears not to cause a problem in England and Wales. However, many herpesviruses persist in their host species without causing overt diseases. It is possible that the virus may continue to persist in UK oyster stocks and only cause disease when conditions are suitable for the virus.

Figure 1. Locations of the three test and reference sites in the Statfjord and German Bight sampling areas, and areas (ICES statistical squares) in which VHSV-positive fish have been captured in or adjacent to the North Sea. Circles: Test areas near the Statfjord oil platform. Diamond: Statfjord reference area. Filled squares: test areas in the German Bight. Triangle: German Bight reference area. Open squares: ICES squares in which VHSV-positive fish have been captured.

Table 2. Species and number of fish sampled at each of three contaminated and one reference site near the Statfjord oil platform and in the German Bight.

Station Species caught Number of fish sampled

Statfjord area 1 Whiting (Merlangius merlangus) 30

Statfjord area 1 Atlantic herring (Clupea harengus) 9

Statfjord area 2 Atlantic cod (Gadus morhua) 2

Statfjord area 2 Whiting 28

Statfjord area 3 Saithe (Pollachius virens) 38

Statfjord area 3 Atlantic herring 2

Statfjord area 3 Whiting 19

Statfjord area 4 (reference) Atlantic herring 50

German Bight 1 Atlantic herring 10 x 5 fish pools

German Bight 2 Atlantic herring 10 x 5 fish pools

German Bight 3 Whiting 50

German Bight 3 Atlantic herring 3 x 5 fish pools

German Bight 4 (reference) Atlantic herring 25

German Bight 4 (reference) Whiting 14

German Bight 4 (reference) Atlantic cod 19

As part of the VINO project we participated in a ring-trial of PCR and in situ hybridisation (ISH) with three other laboratories in which 30 samples per trial were tested; the samples were different in each trial. Two primer pairs were used in the PCR trial, and three of the four laboratories obtained similar results to each other with both pairs. The results from the fourth partner contained a large number of false-positives. The results obtained from the ISH trial were more variable between the participants, which in part reflected the inexperience of some of them with that technique. Our results were quite similar to the expected results, although we did have two false negative results.

In situ hybridisation (ISH). Work commenced on developing the ISH method for use with a number of rhabdoviruses. Initially the work focused on VHSV and infectious haematopoietic necrosis virus (IHNV), but later the emphasis changed to the cyprinid rhabdoviruses (spring viraemia of carp [SVCV]-like viruses). The ISH method is illustrated in Figure 2.

Figure 2. Schematic representation of the ISH method

The technique is typically applied to histological sections of tissues, although it can be done on tissue imprints or monolayers of cell cultures. The basis of the method is as follows. A DNA probe specific for a small portion of the nucleic acid of the virus (or other pathogen) is constructed, and labelled with digoxigenin, a protein. The tissues are first treated with a proteinase enzyme, to make them more permeable to the reagents. The probe is then added and the nucleic acid in the cells is denatured by heating, which allows the probe to hybridise to any complementary sequences (i.e. specific sequences of the virus of interest) in the cells. The probe will not hybridise in the absence of the complementary sequences. Excess or non-hybridised probe is washed away, then specific rabbit antibody against digoxigenin is added, which binds to the digoxigenin. Excess rabbit antibody is washed away. An enzyme-labelled second antibody, directed against the rabbit antibody is added, which binds to any rabbit antibody present. Following another wash, a colourless substrate of the enzyme is added, which in the presence of the enzyme changes colour – in this case to a blue colour. Cells that contained the virus will therefore appear blue when observed under the microscope.

Nucleic acid probes were produced against VHSV, and an ISH method developed. Assessment of the probes and method was done under Objective 2.

Nucleic acid probes were produced against representatives from each of the four genogroups of cyprinid rhabdoviruses (SVCV-like viruses). Tests by Southern blotting demonstrated a strong homologous reaction, but also a lesser heterologous cross-reaction with two probes. The probes were used for ISH on infected cell cultures, and again there was a strong positive homologous reaction, and any heterologous reactions were very weak. ISH on cell cultures can be used to distinguish between the isolates. The use of reverse hybridisation appeared to hold promise as a suitable method to distinguish between the four genogroups, but it

Digoxigenin-labelled DNA probe specific for the viral nucleic acid

Key: Non-infected cell

Rabbit antibody against digoxigenin

Goat anti rabbit antibody conjugated with alkaline phosphatase

Colourless enzyme substrate BCIP/NBT

Alkaline phosphatase hydrolyses BCIP/NBT to a blue product. Cells containing specific virus nucleic acid turn blue

was felt that the development of that method was more appropriate under Defra contract F1152, and so the work was done under that project.

The emphasis of the development and validation of in situ hybridisation (ISH) methods for VHSV and IHNV changed to using SVCV as a model rhabdovirus following the isolation of SVCV in the USA in summer 2002. This laboratory was fulfilling its role as Organisation International des Epizooties (OIE) (World Organisation for Animal Health) reference laboratory for SVCV under this contract, and provided assistance to the US authorities. The work on the ISH method for SVCV was given a higher priority in order to analyse archive fixed material taken from carp mortalities in the US over several years. The ISH showed the presence of SVCV in material fixed in 1989 (Figure 3), indicating that SVCV had been present in the USA at that time.

Figure 3. ISH applied to kidney tissue taken from a moribund carp in the USA in 1989. Blue stained cells are positive for SVCV RNA.

However, further information on the 1989 SVCV was obtained by another method being developed, which was called archive PCR. That method allowed RT-PCR analysis of archive fixed tissues. Essentially thick sections are cut from histological blocks of fixed, wax-embedded tissues, the wax is removed and the nucleic acid is extracted from the tissues. The standard PCR or RT-PCR can then be used to look for viral nucleic acid in the sample. The method has the benefit of allowing nucleotide sequencing on positive material. The US archive samples were positive by RT-PCR as well as by ISH, and subsequent nucleotide sequence analysis showed that the sequence was similar to that present in SVCV isolates of European origin, whereas the sequence of the 2002 isolates were similar to that present in SVCV isolates of Asian origin. This meant that although SVCV was present in the US in 1989, the later outbreaks were not caused by that SVCV type, but were caused by a SVCV type from a different source, and was possibly a more recent introduction. The identification of SVCV in the archive material led the US authorities to reconsider their approach to follow-up action and monitoring studies. The immunoassay for SVCV antibody in fish developed at CEFAS in an earlier contract was used in epidemiological studies to determine the spread of infected fish in the US. All carp sampled from Cedar Lake (the site of one of the two separate US outbreaks) were SVCV-antibody positive, and blood samples from carp from several sites on the Mississippi, which is fed from Cedar lake, were also SVCV antibody positive. This provided valuable information for the proposed control measures. The archive PCR will only be successful if the tissues are not over-fixed i.e. kept in the fixative for a prolonged time, as that can cause degradation of the nucleic acid, particularly RNA. The method for SVCV and KHV in carp tissues was further refined by designing a beta actin primer pair for carp. The presence of a product following the PCR using that primer pair shows that the tissues were not over-fixed and hence are suitable for archive PCR to search for viral nucleic acid. This primer combination will also indicate genomic DNA contamination of RNA (by size of product).Nucleic acid probes have been designed and produced against IHNV, but their full assessment for their suitability for use in blotting and ISH reactions was not completed because of other priorities.

Objective 2. Bream rhabdovirus.

Please note that genetic studies have shown that many rhabdovirus isolates from cyprinids and coarse fish (including the bream rhabdovirus) are related and are classified in the same Genogroup., which has provisionally been called the tench rhabdovirus (TeRV) Genogroup, after the first virus isolated that was assigned to the group. Also note that many of the isolates are called TeRV, even though they may have been isolated from different hosts, as the viruses have a wide host range. The nomenclature of these viruses needs amending, but the current nomenclature will be used here.

Five of six sites found positive for the bream rhabdovirus in 1999 were re-sampled in 2000. No virus was isolated in cell culture and no tissue was positive by the PCR except for samples of blood cells from two fish from one site. ISH assays were carried out on fish tissue samples from that site, and 16 of 30 fish were positive at a low level. No specific antibody was found in blood samples taken from bream exposed to the virus.

In 2004 tench rhabdovirus (TeRV) was isolated during a disease outbreak in barbel at the Environment Agency’s Calverton fish farm. In subsequent investigations the virus was also isolated from roach and a competitive ELISA was used to detect antibodies to TeRV in serum samples taken from a range of cyprinid fish species held on the site. Antibodies to TeRV were detected in barbel and roach and were also detected in crucian carp and chub. The majority of the fish species on the fish farm are reared from wild-caught broodfish. The competitive ELISA has good potential as a broodstock screen for exposure to TeRV.

Objective 3. Impact of emerging pathogens.

Koi herpesvirus. During 1998, cyprinid-fish farm sites in Israel that regularly supply fish for the world-wide ornamental trade suffered high mortalities among their common and koi carp stocks. Severe gill necrosis was the most distinctive and consistent pathological sign among the affected carp. Mortalities with a similar aetiology were also seen in ornamental carp populations in Germany, Holland, Belgium, United Kingdom and the USA. A herpesvirus (koi herpesvirus, KHV) was isolated from koi carp in the USA and in Israel. The virus is very tissue culture cell-line specific and was first isolated in koi fin (KF-1) cell cultures. That cell line was initiated in the USA, and we obtained it for disease diagnostic and research purposes. On two occasions in 2000, KHV was isolated using the KF cells from koi carp imported into the UK from Israel and Malaysia. In 2001, an additional outbreak of disease occurred in koi carp that had been recently imported into the UK from Israel. Although those fish exhibited typical symptoms of the KHV infection and KHV DNA could be amplified by PCR from fish tissues, the virus was not isolated in tissue culture. A number of techniques were used in an attempt to improve the isolation of KHV. Since 2001 an increasing number of cases of KHV were diagnosed and over the course of this contract research on the diagnosis, identification and characterisation of the virus was undertaken.

Diagnosis and identification. In vitro growth enhancing methods (pre-treatment of cell monolayers with PEG 6000 and supplementing the virus culture medium with low concentrations of trypsin) were used to try to increase the yield of virus in KF cells, but without success.

We were fortunate to be provided with pre-publication data on a PCR for the virus (Gilad et al. 2002) which was modified and used in comparison with the virus isolation method as a means of diagnosing the disease. Analysis of the data from 270 samples from 49 batches of fish showed that the PCR was more sensitive than virus isolation, particularly on samples of viscera. The results of the comparisons are as follows: 39.9 % of gill samples were positive by virus isolation and 46.4% were positive by PCR; 17.9% of viscera samples were positive by virus isolation and 35.7% were positive by PCR. The detection of the virus in gill samples and visceral samples from the same fish were compared, and in 26.8 % of the comparisons by cell culture and 10.7 % of the comparisons by PCR the virus was detected in only one of the two sample types. Hence both types of tissue should continue to be taken for diagnostic purposes. The PCR is now used routinely at the laboratory, but still alongside the virus isolation method. A further increase in sensitivity of the PCR method was obtained by the development of a nested PCR. However, the greater sensitivity has meant that more stringent precautions to prevent false-positives are necessary. All PCR assays used for diagnostic purposes require a confirmatory test, which can be nucleotide sequencing when a small number of samples are processed. However, as there were a large number of KHV samples we have developed a Southern blotting technique that can also be used as the confirmatory test. A labelled KHV-specific probe confirms that the amplified PCR fragment is not only of the expected size, but also corresponds to the expected sequence in the KHV genome.

Archive PCR and ISH methods for the detection of KHV DNA have been developed. Six KHV primer sets were compared for use in the PCR on archive material. One primer set was selected and validated on a range of

sample material. The primers were used to analyse archive material from a disease outbreak in 1996 affecting koi, mirror and common carp held in polyculture with other non-affected species. KHV DNA was identified in the koi carp, and the finding was confirmed by ISH. That result is significant as it means that KHV was present in this country before the problems with KHV disease were recognised in Israel and other countries from 1998 onwards. The virus was not isolated in 1996 as we now know that the cell cultures used for virus isolation at that time do not support the replication of KHV. The archive-PCR has been successful in detecting and confirming KHV DNA in common carp tissues sampled by the Environment Agency (EA) during suspected occurrences of spring carp mortality syndrome in 1999.

Another significant finding was the detection of KHV DNA by PCR, confirmed by ISH in common carp sampled during disease outbreaks with high mortalities at a number of fishery sites in England in 2003. Prior to that, the virus had only been isolated from koi carp, and usually only at dealers’ premises, garden centres, or koi hobbyists’ ponds. Virus was not isolated in cell culture from the initial disease outbreaks, but it was isolated from diseased carp in outbreaks later in 2003, and also in 2004. The isolate was shown to be genetically similar to KHV isolated from koi and was shown to be comparable in virulence (75-90% mortality) in virus-challenge studies in carp.

Antibody detection. Development of an enzyme-linked immunosorbent assay (ELISA) for detecting antibody to KHV (KHVabEIA) in carp serum was also carried out. Serum samples from carp exposed to KHV gave high absorbance values in the ELISA at dilutions of 1:3200 and above. The criteria used for a fish to be considered positive for KHV antibodies was an absorbance reading at the 1:1600 dilution that was greater than the average for the negative control plus three times its standard deviation, and if all other lower dilutions had higher absorbance readings than the previous dilution. The antibody response was then used to monitor for KHV in a population of carp that had been exposed to KHV, when cell culture and PCR techniques were not reliable at detecting virus in sub-clinical fish. Carp were sampled monthly for nine months, and antibodies were detected at each sampling occasion with a prevalence of between 20 and 40%. All non-KHV-exposed fish tested were negative for KHV-specific antibodies by ELISA.

The KHVabEIA has potential as a screening test of exposure to the virus in carp but needs to be validated to ensure reliability and specificity. As a first step in this validation an ELISA was developed to detect antibodies in carp serum to the cyprinid herpesvirus (CyHV-1) that causes carp pox. Serum from carp exposed to CyHV-1 were tested in the ELISA (CHVabEIA) and shown to contain antibodies to CyHV-1. These same sera did not cross-react in the KHVabEIA and the KHV antibody positive sera did not cross-react in the CHVabEIA. However, further validation is needed with serum samples from wild common carp. It is possible that some populations of common carp may have been exposed to CyHV-1 and KHV.

Comparative studies. The nucleotide sequences of a 484 base pair PCR product obtained from the first UK isolates showed 100% identity to KHV isolated in the USA. This means that the virus either has a very conserved genome, or the same virus has been spread to different countries; the latter was most likely for these isolates, as both were isolated from koi carp imported from Israel.

Short portions (400 bp) of the genome of 13 KHV isolates have been sequenced, and one such sequence was found to be variable. That variation is associated with different geographic origins of the isolates (Asia and Israel), and also differs with year of origin of some isolates. Thus there are genertic differences between some isolates, and there may be useful markers for epidemiological studies, but further work is needed to confirm this finding.

Approximately 50 kbp of the KHV genome (30% of the total genome based on a genome size of 150kbp) has been sequenced and analysed. A large number of putative genes have been identified, many of which, share significant homology with genes found in channel catfish virus (CCV) and other herpesviruses. The most relevant are two neighbouring KHV genes in a 7.4 kbp fragment that share significant homology with the CCV helicase (ORF 25) and capsid proteins (ORF 27). The evolutionary relationship for the other genes in this region remains speculative, but since the overall gene arrangement for this region is also similar to the arrangement seen in the CCV genome, it strongly indicates that KHV and CCV are related herpesviruses. These data, combined with data from other laboratories have allowed a comparison with the DNA of other herpesviruses isolated from cyprinids. KHV DNA is different from (CyHV-1), associated with skin papillomas, and cyprinid herpesvirus 2 (CyHV-2), originally isolated from goldfish, and it has been proposed in a paper accepted for publication that KHV be recognised as cyprinid herpesvirus 3.

Antiviral molecules in carp (EU AVINSI project QLRT-2001-01691). Different fractions were extracted from tissues of carp infected with KHV to test for anti-viral activity. The extractswere sent to one of the other partners for analysis, which is ongoing.

RNA was purified from virus-infected and control leucocytes to search for virus induced genes. Head kidney leucocytes from common carp were purified by centrifugation on Percoll®, and were seeded into cell culture flasks. Each day for 5 days the cells were infected with KHV, such that they were exposed to the virus for 16, 24, 48, 72, and 96 h. Control flasks were harvested and pooled and the infected flasks were also harvested and pooled. Total RNA was extracted from each pool using Trizol®. An aliquot was sent to another partner for detection of inhibitor of apoptosis genes, and the remainder was used to prepare subtracted cDNA libraries enriched in virus-induced genes.

cDNA was synthesised from each RNA preparation of the primary leucocyte cell culture using a cDNA Synthesis kit. Suppressive Subtractive Hybridisation was then performed using a PCR Select cDNA Subtraction Kit. Forward and reverse subtractions were performed as well as preparation of non-subtracted cDNA for later use in screening. The efficiency of subtraction was analysed using primers for the virus activated cellular genes interferon and Mx. During the PCR, samples were removed after 18, 23, 28, and 33 cycles and were size separated on a 2% agarose gel. The results indicated a product for carp interferon was visible from the unsubtracted DNA control only after 33 cycles whereas the subtracted cDNA produced a visible product after 23 cycles indicating a 40 fold enrichment, as a difference of 5 cycles equates approximately to an enrichment of 20 fold. Products for the Mx gene were visible after fewer cycles from both subtracted and unsubtracted cDNAs but a lag of approximately 5 cycles was seen in the unsubtracted cDNA again indicating enrichment of the subtracted library.

The PCR product of the forward subtraction was ligated into pGEM-T vector and transformed into JM109 cells. Four hundred and twenty individual colonies have been picked, grown overnight and checked for insert by PCR amplification. The PCR products of these clones have been spotted onto membranes and are being screened for virus induced genes using a PCR Select Differential Screening Kit. Prior to screening with the subtracted and non-subtracted forward and reverse library probes membranes are being probed with digoxigenin-labelled random primed purified KHV DNA probe. Clones indicating a KHV origin by hybridisation with KHV probe will be eliminated from the screening process. Initial probing resulted in high intensity signals for all spots on all membranes with all probes. No differences between unsubtracted probes and subtracted probes were detectable. Further probing experiments were performed to attempt to obtain differential expression results with the various probes. However, to date no significant differences have been seen in spot intensity on duplicate membranes when probed with the four different probes. Thus we have not yet isolated differentially expressed sequences from the library. However since a variety of PCR checks have demonstrated the library is of good quality and has been successfully subtracted for common genes and enriched for certain up-regulated genes, it must be assumed the problems still lie in the library screening protocol. Further experiments and alterations are ongoing to improve the library screening protocol under the EU-funded component of the AVINSI project.

Asian isolates of SVCV. SVCV is not considered to be an emerging pathogen, but in recent years SVCV strains were isolated that differed in in vitro properties from the previous SVCV isolates. All those newer strains had a connection with fish from Asian countries, and were termed “Asian SVCV”. Some of them were isolated from illegal imports of fish. Further work was done on those strains as emerging diseases. Work in collaboration with Defra contract F1118 identified four Genogroups of vesiculoviruses, one of which contained all the SVCV isolates. The SVCV Genogroup itself comprised four subgroups. The isolates of Asian origin were all classified in subgroup 1a, which also included the isolates from USA. It can be speculated that the USA isolates may have been imported with fish from Asia. The other subgroups (1b, 1c and 1d) comprise isolates from Europe and countries of the former USSR; subgroup 1d contains the largest number of isolates from our culture collection, and comprises isolates predominantly from Europe.

Commercial reagents for identifying SVCV (an ELISA kit, and a monoclonal antibody [MAb] based fluorescent antibody test) were evaluated against 17 representative viruses from the four SVCV subgroups and seven isolates from the three other vesiculovirus Genogroups. The SVCV isolates were selected from distinct branches on a neighbour joining distance tree within each subgroup. The ELISA detected all isolates, but could not discriminate between SVCV and the non-notifiable viruses from the other three vesiculovirus Genogroups. In an initial test the MAb detected 4 of 5 isolates from SVCV subgroup 1d, but no isolates from the other SVCV subgroups, nor from the other vesiculovirus Genogroups. To investigate that further, two further subgroup 1a isolates from adjacent branches of the neighbour joining distance tree were tested by both methods. The ELISA again detected both isolates, but the MAb only detected one of the two isolates. Both of the SVCV isolates not detected by the MAb were isolated from neighbouring countries near Russia (Moldova and Ukraine), whereas the other subgroup 1d isolates that were detected by the MAb were isolated in England, or Yugoslavia. Representatives from the four vesiculovirus Genogroups clearly have common epitopes, but certain isolates can be distinguished by the use of different serological tests. The subgroup 1a isolates can be distinguished from the other SVCV isolates by neutralisation assays with antisera raised against subgroup 1d isolates, and many of the subgroup 1d isolates can be distinguished from the other SVCV isolates with the

MAb. With a range of antisera or Mabs it may be possible to undertake sero-epidemiological tracing studies on new SVCV isolates.

Spring carp mortality syndrome (SCMS). Studies were carried out with SCMS-affected carp in collaboration with the EA. Fish were sampled by EA staff from suspected SCMS mortalies at two lakes and a fishery in 2003. Fish were transported to this laboratory for further investigation. The carp from different sources were kept in different tanks and the temperature of the water was gradually raised from 11 to 16°C, and then up to 21°C, but no mortalities were observed. KHV DNA was detected in one of the original SCMS fish by a nested KHV PCR assay. A slow-growing agent, which appears to be a herpes-like virus by electron microscopy, was isolated in KF-1 cells from another of the SCMS fish. Analysis by PCR has shown that it is not KHV, and, unlike KHV, grows in Epithelioma papulosum cyprini (EPC) cells. PCR analysis has also shown that it is not cyprinid herpesvirus 1 nor cyprinid herpesvirus 2. Unfortunately, that virus was isolated toward the end of this contract and its characterisation is unfinished. Any further work on it will have to be carried on in a new contract.

Examination of histological sections from diseased carp revealed the presence of bacteria in some gill tissues from exposed fish. In other samples of gill tissue areas of necrosis were evident that appeared not to be associated with the presence of a pathogen.

Herpesviruses. Further characterisation work was carried out on herpesviruses isolated in 2003 and 2004 during investigations of disease outbreaks in common carp, koi carp, and goldfish. Two of the herpesviruses from koi carp have been identified by partial gene sequencing and found to be similar to CyHV-1. The virus isolated from goldfish has been shown to be similar to CyHV-2, which has been isolated from goldfish in Korea, Australia and the USA.

Unidentified virus, possible myxovirus. Since 1996 a slow-growing virus has been isolated from diseased carp on numerous occasions in EPC and/or KF-1cells. The cytopathic effect is one of cell fusion forming syncitia. Viruses producing similar cytopathic effects have been described by Body et al. (2000) and Neukirch and Kunz (2001), and in both reports the viruses were described as paramyxovirus-like. However, work on characterising the viruses isolated here has been slow because they grow only slowly in cell culture, and their characterisation was not a high priority compared to other work in this contract. However, the frequency of their isolation has increased in recent years, and there is concern that they may interfere with the isolation of other viruses. A virus producing the same type of cytopathic effect was isolated in 2003 and grew well in KF cells, and further work was undertaken with that isolate. It was visualised by electron microscopy and shown to be similar to the paramyxovirus-like viruses described in the literature. However, on a morphological basis the virus could be an orthomyxovirus or a paramyxovirus, but determination of the number of strands of RNA would distinguish between the two virus types. Isolates from 1996 have been recovered from frozen storage and have been shown to be morphologically similar to the 2003 isolate by electron microscopy. Attempts have been made to purify the fast growing 2003 isolate, but with limited success. One harvest from a sucrose density gradient was analysed on polyacrylamide gels for its polypeptide composition, and the nucleic acid was analysed. However, there was insufficient virus following the purification schedule to resolve either polypeptides or nucleic acid. Studies on the genome of the virus in collaboration with the University of Munich have commenced.

Irido-like virus. During investigations into the TeRV outbreak at Calverton fish farm an irido-like virus was isolated from apparently healthy dace sampled at the site. Transmission electron microscope images have been obtained and the virus morphology strongly indicates an iridovirus. As the iridovirus EHNV, which has a wide host range is notifiable to the OIE, tests were done to determine whether the dace isolate was EHNV. Cell culture harvests were tested in a PCR assay for systemic iridoviruses (ranaviruses) and found to be negative. Reference ranavirus isolates from catfish and perch were positive in the assay, and so the dace isolate is not EHNV. Further work is needed to identify the virus and complete further genetic characterisation. This will be done in new Defra project FC1156.

Aquabirnaviruses. Aquabirnaviruses isolated in Singapore from imported fish have been shown to form a new (fourth) birnavirus Serogroup. This is significant from an international basis for identification of related isolates that may be isolated.

Objective 56. Fish waste.

As a result of new legislation, accepted methods of disposal of fish mortalities such as burial with a covering of lime are no longer permitted, but approved methods of disposal of fish by-products were either not available UK-wide, or expensive. Disposal of fish by-products by composting or ensiling are permitted under the legislation, but there are very few data on the survival of fish pathogens under those conditions. Defra

requested that such data be obtained through this contract with the highest priority, in conjunction with work to be carried out by colleagues at the FRS Laboratory in Aberdeen. Research was also done under other Defra contracts (which included survival data for bacteria), and the full results of all the Defra-funded work can be found in the Final Report of Defra contract F1157.

The aim of the work was to determine the most resistant virus to ph 4 (ensiling conditions) and a temperature of 60°C (the minimum temperature likely for composting. Subsequently the survival of viruses at pH 12 was also determined. The two laboratories agreed protocols for the work and conducted calibration trials to ensure that the results from both laboratories were truly comparable. This was essential as each laboratory was to test different pathogens, and then combine the data to select the most resistant virus to each test parameter. For the calibration trial each laboratory tested the survival of infectious pancreatic necrosis virus (IPNV) under the three test parameters at different time intervals. It was agreed that parity between the results would be obtained if the values obtained by each laboratory for the reduction in titre at any time point were within 1 log10 unit of each other. Parity was achieved and the two laboratories then tested different viruses. This laboratory tested IPNV serotypes A2 (Sp), A3 (Ab), and B1, SVCV from genogroup Ia (Asia) and genogroup Id (Europe), channel catfish virus (CCV), and European iridoviruses (from catfish and from sheatfish). The FRS laboratory tested VHSV, IHNV, ISAV and viral encephalopathy and retinopathy virus. The viruses were exposed to 60°C, pH 4 and pH 12 for different time intervals and the most resistant viruses were determined for each parameter. The results of the inactivation studies for this laboratory are summarised in Table 8.

Table 8. Summarised results of the virus inactivation trials

PathogenViability of viruses following exposure to test parameters60°C pH 4 pH 12

IPNV Sp100% inactivation within 24 h

Survived 28 days, losing only 0.22 log10

units of infectivity

100% inactivation within 20 min

IPNV AbSurvived 24 h, losing 2.8 log10 units of infectivity

Survived 28 days, losing only 0.33 log10

units of infectivity

100% inactivation within 1 h

IPNV 970160

100% inactivation within 24 h

Survived 28 days, losing less than 0.2 log10 units of infectivity

100% inactivation within 1 h

SVCV D120

100% inactivation within 24 h

Survived 28 days, losing 1.7 log10 units of infectivity

Titre reduced by nearly 5 log10 units of infectivity after 6 h, but 3 x 103

TCID50 surviving

SVCV 880062100% inactivation within 24 h

Survived 28 days, losing 2.1 log10 units of infectivity

100% inactivation within 6 h

EHNV (sheatfish)

100% inactivation within 24 h

Survived 28 days, losing 0.63 log10 units of infectivity

Titre reduced by over 4 log10 units of infectivity after 6 h, but 3.8 x 103

TCID50 surviving

EHNV 562/92

100% inactivation within 24 h

Survived 28 days, losing 0.93 log10 units of infectivity

Titre reduced by over 4 log10 units of infectivity after 6 h, but 1.9 x 103

TCID50 surviving

CCV

100% inactivation within 24 h

100% inactivation within 24 h

Titre reduced by over 2 log10 units of infectivity after 6 h, but 3.1 x 103

TCID50 surviving

IPNV Ab was most resistant to 60°C, and SVCV D120, iridoviruses and CCV were contenders for the most resistant viruses to pH 12. Only CCV was completely inactivated by pH 4, and the other viruses survived for 4 weeks at that pH. Once all the inactivation results had been obtained by both laboratories the results were compared to determine the most resistant pathogens to 60°C and pH 12. As several viruses survived for 4 weeks at pH 4, it was agreed that no further work would be done at that pH. From the combined results the most resistant virus to 60°C was IPNV Ab and to pH 12 was ISAV. Those viruses were then used in field trials of the composting and alkaline hydrolysis procedures. The results of those trials are reported in the Final Report of Defra contract F1157.

Other international collaborations through this contract.

EC project EUROPA – Development of a European resource to assist phylogenetic analysis of important fish pathogens (including SVCV).

Follow-up studies on the SVC disease outbreak in North Carolina, USA. Serum antibody tests on wild cyprinid fish species. In collaboration with US Fish & Wildlife Service ; APHIS, USDA.

Further investigations of carp fish kills in Wisconsin and the Mississippi river basin prior to the SVC outbreak in Cedar Lake, Wisconsin. Analysis of preserved archive carp tissues for presence of SVCV. In collaboration with State of Wisconsin, Department of Natural Resources and University of Wisconsin-Madison.

Identification and sub-genogrouping of SVC virus isolates from Belgium in collaboration with Division of Research and Guidance on Pisciculture, Centre d’Economie Rurale, Marloie, Belgium.

Identification of putative SVC viruses isolated from Rainbow trout in Russia in collaboration with the All-Russia Research Institute of Freshwater Fisheries, Laboratory of Ichthyopathology, Rybnoe, Moscow province, Russia.

Meetings organised

Staff funded by this project helped organise and participated in the following:

Workshop on Diagnostic Techniques for Fish Diseases with Special Emphasis on Carp held in Weymouth in June 2003, co-sponsored by Defra and the EU.

International Workshop on Koi Herpesvirus held in London in February, 2004, sponsored by Defra.

Conclusions:

This work has provided support for statutory diagnostic and control measures; helping to ensure high standards of animal health, and has helped to protect and improve the environment, conserve and enhance biodiversity, by:a) Enhancing our ability to rapidly and unequivocally identify notifiable viruses.b) Providing information on the (negative) distribution of ISAV in England and Wales for disease control

purposes.c) Providing information on the significance of new and emerging diseases (e.g. bream rhabdovirus, KHV) for

consideration as to whether the diseases should be notifiable, and their potential impact on wild fish.d) Providing methodologies to enable us to continue to provide an efficient and effective diagnostic service,

and helping maintain our high health status.e) Providing information to enable us to comply with EU regulations (survival of pathogens in animal by-

products following ensiling or composting).

Future need for R&D: There is a need for work in the following areas:a) Continuation of studies to determine the significance of notifiable and new and emerging diseases on

native fish stocks e.g. possible paramyxovirus affecting carp.b) Development of new cell lines to enhance our diagnostic capability.c) Determine whether survivors of KHV become virus carriers. Development of non-lethal sampling methods,

and detection of carrier fish by specific antibody detection methods (KHV, SVCV, and others).d) Development/assessment of methods to enable us to use measurements of the (cellular) immune

response of fish to detect carrier fish.

References

Body,A., Lieffrig,F., Charlier,G., & Collard,A. (2000). Isolation of virus-like particles from koi (Cyprinus carpio) suffering gill necrosis. Bulletin of the European Association of Fish Pathologists, 20: 87-88.

Gilad,O., Yun,S., Andree,K.B., Adkison,M.A., Zlotkin,A., Bercovier,H., Eldar,A., & Hedrick,R.P. (2002). Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi. Diseases of Aquatic Organisms, 48: 101-108.

Mjaaland, S., Rimstad, E., Falk, K. and Dannevig, B.H. (1997). Genomic characterisation of the virus causing infectious salmon anaemia in Atlantic salmon (Salmo salar L.): an orthomxyo-like virus in a teleost. Journal of Virolology, 71, 7681-7686.

Neukirch,M., & Kunz,U. (2001). Isolation and preliminary characterization of several viruses from koi (Cyprinus carpio) suffering gill necrosis and mortality. Bulletin of the European Association of Fish Pathologists, 21: 125-135.

OUTPUTSN.B. The outputs are placed in this part of the form as they will not fit in the next section of the electronic form

Publications: Journals

Betts, A.M., Stone, D.M., Way, K., Torhy, C., Chilmonczyk, S., Benmansour, A. and Kinkelin, P. de (2003). Emerging vesiculo-type virus infections of freshwater fishes in Europe. Diseases of Aquatic Organisms. 57: 201-212.

Chew-Lim, M., Ngoh, G.-H., Chong, S.Y., Chang, S.F., Kueh, S.L.F, Way, K. & Dixon, P.F. (2002). Birnaviruses of potential new serogroups isolated from tropical fish. In C.R. Lavilla-Pitogo and E.R. Cruz-Lacierda, (Eds.) Diseases in Asian Aquaculture IV 219-233. Fish Health Section, Asian Fisheries Society, Manila.

Dikkeboom, A.L., Radi C., Kurth K.T., Marcquenski, S., Engel, M., Goodwin, A.E., Way, K., Stone, D.M. and Longshaw, C.B. First report of spring viremia of carp virus in feral common carp (Cyprinus carpio) in North America. Journal of Aquatic Animal Health 16: 169-178

Dixon, P.F., Avery, S., Chambers, E., Feist, S.W., Mandhar, H., Parry, L., Stone, D.M., Strømmen, H.K., Thurlow, J.K. & Way, K. (2003). Four years of monitoring for viral haemorrhagic septicaemia virus in marine waters around the United Kingdom. Diseases of Aquatic Organisms 54: 175-186.

Gilad, O., Yun, S., Adkison, M.A., Way, K., Willits, N.H., Bercovier, H. & Hedrick, R.P. (2003). Molecular comparison of isolates of an emerging fish pathogen, koi herpesvirus, and the effect of water temperature on mortality of experimentally infected koi. Journal of General Virology 84: 2661-2668.

Haenen, O.L.M., Way, K., Bergmann, S.M. and Ariel, E. (2004). The emergence of koi herpesvirus and its significance to European aquaculture. Bull. Eur. Ass. Fish Pathol. 24: 293-307.

Rowley, H., Graham, D.A., Campbell, S., Way, K., Stone, D.M., Curran, W.L. and Bryson, D.G. (2001). Isolations and characterisation of rhabdoviruses from wild common bream (Abramis brama) and roach (Rutilus rutilus) and from farmed brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) in Northern Ireland. Diseases of Aquatic Organisms 48: 7-15.

Stone, D.M., Ahne, W., Denham, K.L., Dixon, P.F., Tsin-yee Lui, C., Sheppard, A.M., Taylor, G.R. & Way, K. (2003). Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups. Diseases of Aquatic Organisms 53, 203-210.

Varvarigos, P. & Way, K. (2002). First isolation and identification of the Infectious Pancreatic Necrosis (IPN) virus from rainbow trout Oncorhynchus mykiss fingerlings farmed in Greece. Bulletin of the European Association of Fish Pathologists, 22 (3) 195-200.

Way, K., Bark, S.J., Davis, C.B., Denham, K.L., Dixon, P.F., Feist, S.W., Gardiner, R., Gubbins, M.J., Le Deuff, R.-M. , Martin, P.D., Stone, D.M. and Taylor, G.R. (2003). Isolation of a rhabdovirus during outbreaks of disease in cyprinid fish species at fishery sites in England. Diseases of Aquatic Organisms 57: 43-50.

Publications: manuscripts submitted / in press

Alonso, M.C., Le Deuff, R.-M. and Dixon, P.F. Studies on the pathogenesis of viral haemorrhagic septicaemia virus in turbot, Scophthalmus maximus. Submitted to Diseases of Aquatic Organisms.

Dixon, P.F. and Longshaw, C.B. Assessment of commercial test kits for identification of spring viraemia of carp virus. Submitted to Diseases of Aquatic Organisms.

Dixon, P.F., Longshaw, C.B., Jones, G.J. and Stone, D.M. (2005) Monitoring for viral haemorrhagic septicaemia virus and infectious salmon anaemia virus at contaminated and reference areas in the North Sea. Journal of

Environmental Toxicology and Chemistry (in press).

Dixon, P.F., Ngoh, G.-H., Chang, S.F., Kueh, S.L.F., Way, K. A fourth aquabirnavirus serogroup. Submitted to Journal of General Virology

Waltzek, T.B., Kelley, G.O., Stone, D.M., Way, K., Hanson, L., Fukuda, H., Hirono, I., Aoki, T., Davison, A.J. and Hedrick, R.P.. (2005) Koi herpesvirus represents a third cyprinid herpesvirus (CyHV-3) in the family Herpesviridae. Journal of General Virology (in press).

Publications: Internal reports/Limited circulation publications

Ariel, E. (editor) (2003). Report of the Workshop on Diagnostic Techniques for Fish Diseases, with Special Emphasis on Carp. CEFAS Laboratory, Weymouth, June 2 – 6, 2003. 25pp.

Dixon, P.F., Bark, S.J., Le Deuff, R.-M. & Martin, P. (2000). Herpesviruses in marine molluscs. Shellfish News, No 10, 18-20

Le Deuff, R.-M. & Richens, L.F.. (2002). Chronic mortality in a bivalve mollusc hatchery: a case study. Shellfish News, 14: 18-20.

Renault, T. (Co-ordinator) (2001). Diagnosis of oyster herpes-like virus: development and validation of molecular, immunological and cellular tools. Second periodic progress report for European Commission contract FAIR CT98-4334.

Renault, T. (Co-ordinator) (2002). Diagnosis of oyster herpes-like virus: development and validation of molecular, immunological and cellular tools. Final report for European Commission contract FAIR CT98-4334.

Renault, T. (Co-ordinator) (2003). Anti-viral innate immunity in cultured aquatic species. First annual report for European Commission contract QLK2-CT-2002-01691.

Renault, T. (Co-ordinator) (2004). Anti-viral innate immunity in cultured aquatic species. Second annual report for European Commission contract QLK2-CT-2002-01691.

Stagg, R. & Hill B. (Co-chairs of survey working group) (2000). GB survey of wild fish for infectious salmon anaemia (ISA). First periodic progress report.

Stagg, R. & Hill B. (Co-chairs of survey working group) (2000). GB survey of wild fish for infectious salmon anaemia (ISA). Second periodic progress report.

Stagg, R. & Hill B. (Co-chairs of survey working group) (2001). GB survey of wild fish for infectious salmon anaemia (ISA). Final report.

Raynard, R.S., Dixon, P.F., Gardiner, R., Gardiner, W.R., Grant, R., Murray, A.G., Longshaw, C., Gregory, A., Quickfall, S., Macdonald, A.I.M., Sheppard, A.M., Cunningham, C.O., Stone, D.M., Bain, N., Taylor, G., Hill, B.J. and Stagg, R.M. (2002) Survey of wild salmonid fish in Great Britain in year 2000 for infectious salmon anaemia (ISA).

Oral & poster presentations at meetings

Alonso, M.C., Le Deuff, R.-M. and Dixon, P.F. (2001). Studies on the pathogenesis of viral haemorrhagic septicaemia virus in turbot, Scophthalmus maximus. X International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Dublin. Abstract P-226.

Alonso, M.C., Le Deuff, R.-M. & Dixon, P.F. (2002). Aplicación del método de hibridación in situ (ISH) en la localización de VHSV en tejidos de peces infectados. IV Reunion de Microbiologia del medio acuatico, Seville. Oral presentation.

Betts, A.M., Kinkelin, P. de, Benmansour, A., Torhy, C., Stone, D.M. and Way, K., (2001). Biochemical and genetic characterisation of a new group of fish vesiculoviruses. X International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Dublin. Abstract P-094.

Dixon P.F., Betts, A., Davis, C., Le Deuff, R.-M., Martin, P. Stone, D., Taylor, G. & Way, K. (2000). Research on the identification, diagnosis and significance of notifiable and emerging virus diseases of fish and shellfish. British Trout Farming Conference, Sparsholt. Poster presentation.

Dixon, P.F., Alonso, M.C., Gregory, A., Le Deuff, R.-M., Longshaw, C., Sheppard, A., Stone, D., Taylor, G. and Way, K. (2001). MAFF project FC1136: Research on the identification, diagnosis and significance of

notifiable and emerging virus diseases of fish and shellfish. Application of new molecular biology methods for detection of fish pathogenic viruses. British Trout Farming Conference, Sparsholt. Poster presentation.

Dixon, P.F., Ngoh, G.-H., Chang, S.F., Kueh, S.L.F. and Way, K. (2001). A fourth aquabirnavirus serogroup. X International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Dublin. Abstract P-256.

Dixon, P.F., Longshaw, C.B., Jones, G.J. and. Stone, D.M. (2002). Monitoring for fish viruses. Biological Effects of Contaminants in Pelagic Organisms, wash-up meeting, Copenhagen. Oral presentation. Abstract p 33.

Dixon, P.F., Marcquenski, S., Le Deuff, R.-M., Denham, K.L., Sheppard, A.M., Steedman, L., Stone, D.M. and Way, K. (2003). Use of in situ hybridisation and the polymerase chain reaction to identify spring viraemia of carp virus and koi herpesvirus in fixed archive material. XI International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Malta. Oral presentation, abstract O-028.

Goodwin, A.E., Dikkeboom, A., Radi, C., Kurth, K., Marcquenski, S., Way, K. and Stone, D.M. (2002). Spring viremia of carp virus (SVCV) in North America. 5th International Symposium on Viruses of Lower Vertebrates, Seattle, Oral presentation. Abstract p 6.

Le Deuff, R.-M., Way, K., Ecclestone, L., Dixon, P.F., Betts, A.M., Stone, D.M., Gilad, O. and Hedrick, R.P. (2001). Development and comparison of techniques for the diagnosis of koi herpesvirus (KHV). X International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Dublin. Abstract P-257.

Le Deuff, R.-M., Alonso, M.C., Dixon, P.F., Ecclestone, L., Longshaw, C.B., Martin, P.D., Peeler, E.J., Steedman, L.C., Stone, D.M. and Way K. (2002). Development and validation of diagnostic techniques for the detection of koi herpesvirus (KHV). XII International Congress of Virology, Paris. Abstract V-634, p 216.

Le Deuff, R.-M., Stone, D. M., Way, K., Martin, P.D. and Dixon, P. F. (2002). Differentiation of serologically related cyprinid rhabdoviruses by molecular genetic methods. Proceedings of the 4 th International Symposium on Aquatic Animal Health, New Orleans. Oral presentation. Abstract p 142.

Le Deuff, R.-M., Stone, D. M., Way, K., Martin, P.D. and Dixon, P. F. (2002). In situ hybridisation : a method for the differentiation of spring viraemia of carp virus from similar fish rhabdoviruses. XI International Congress of Virology, Paris. Abstract V-635, p216.

Richens, L.F., Le Deuff, R.-M., Bayley, A.E., Martin, P.D. and Thomas, K.V. (2001). Chronic mortality in a bivalve mollusc hatchery: a case study. X International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Dublin. Abstract P-206.

Sheppard, A.M., Le Deuff, R.-M., Dixon, P.F., Way, K. and Stone, D.M. (2002). The detection and differentiation of piscine vesiculo-like viruses using an RT-PCR reverse hybridisation technique. XI International Congress of Virology, Paris.

Sheppard, A.M., Le Deuff, R.-M. and Stone, D.M. (2003). The use of reverse hybridisation to discriminate between piscine vesiculo-type viruses and to identify SVCV subgroups. XI International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Malta. Poster presentation, abstract P-060.

Steedman, L.C., Joiner, C.L., Le Deuff, R.-M. , Longshaw, C.B., Stone, D.M., Way, K. and Dixon, P.F. (2003). Development of a polymerase chain reaction-based assay for the detection of koi herpesvirus DNA in formalin fixed, wax embedded tissues. XI International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Malta. Poster presentation, abstract P-181.

Stone, D.M., Betts, A.M., Dixon, P.F. and Way, K. (2000). The marine viral haemorrhagic septicaemia virus: does it pose a threat to farmed rainbow trout, Oncorhynchus mykiss? British Trout Farming Conference, Sparsholt. Poster presentation.

Stone, D.M., Dixon, P.F., Le Deuff, R.-M., Sheppard, A. and Way, K. (2002). Studies on the pathogenicity of marine viral haemorrhagic septicaemia virus for rainbow trout (Oncorhynchus mykiss) using reverse genetics techniques. XI International Congress of Virology, Paris. Oral presentation. Abstract V-116, p 40.

Stone, D.M., Dixon, P.F., Longshaw, C.B., Sheppard, A.M. and Way, K. (2002). Phylogenetic analysis of partial glycoprotein gene sequences identifies a number of distinct sub-groups of spring viraemia of carp virus. 5th International Symposium on Viruses of Lower Vertebrates, Seattle. Oral presentation. Abstract p 7.

Way, K., Le Deuff, R.-M., Ecclestone, L., Feist, S.W., Dixon, P.F., Wildgoose, W.H. and Hedrick, R.P. (2001) Isolation of a herpesvirus during disease outbreaks in adult koi carp, Cyprinus carpio, in the UK. X

International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Dublin. Abstract O-129.

Way, K., Denham, K.L., Dixon, P.F., Longshaw, C.B., Sheppard, A.M. and Stone, D.M. (2003). Atypical spring viraemia of carp virus (SVCV) isolated from carp imported into the UK: evidence from phylogenetic analysis of partial gene sequences for a number of distinct sub-groups of SVCV. XI International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Malta. Poster presentation, abstract O-079.

Oral presentations at meetings as invited speaker

Dixon, P.F., Haenen, O.L.M., Beevers, N., Denham, K.L., Joiner, C.L., Longshaw, C.B., Le Deuff, R.-M., Paley, R., Stone, D.M., St-Hilaire, S. and Way, K. (2004). Status of koi herpesvirus disease in Europe, and research on the virus in the United Kingdom. Special Symposium: KHV infection: present status and future prospects for prevention. Tokyo University of Marine Science and Technology, Tokyo, Japan. December 2004

Way, K., Denham, K.L., Dixon, P.F., Longshaw, C.B., Sheppard, A.M. and Stone, D.M. (2003). Atypical spring viraemia of carp virus (SVCV) isolated from carp imported into the UK: evidence from phylogenetic analysis of partial gene sequences for a number of distinct sub-groups of SVCV. 28th Eastern Fish Health Workshop, Gettysburg, PA, USA.

Way, K. , Le Deuff, R.-M., Stone, D.M., Denham, K.L., St-Hilaire, S. (2004). Koi herpesvirus: diagnostics and research at CEFAS Weymouth laboratory 2000-2003. International Workshop on Koi Herpesvirus, London, February 2004.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.