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 (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 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.

CSG 15 (1/00) 3 Project Research on viruses of fish and shellfish MAFF FC1105 title project code

Scientific report (maximum 20 sides A4)

The scientific objectives of this project were:

1 To investigate new virus isolates which are intermediate between spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFR) with the view to being able to identify them as either one or the other virus, or to propose a re-classification of the SVCV/PFR complex.

2. To undertake virulence studies on the UK viral haemorrhagic septicaemia virus (VHSV) isolates (with particular regard to host species) and compare them to other VHSV isolates of known virulence or avirulence. To determine whether there are any markers of virulence/avirulence (in conjunction with DP0105)

3. To continue with the production of monoclonal antibodies (MAbs) against VHSV, viral haemorrhagic septicaemia virus (IHNV) and SVCV. To prepare antisera against other viruses as necessary.

4. To identify novel viruses isolated during diagnostic investigations.

5. To become familiar with the isolation/identification of exotic viruses.

6. To monitor the use of the antibody screening test.

7. To assess new diagnostic tests as appropriate.

8. To maintain a knowledge base of virus diseases of fish and shellfish.

These objectives have been met in full, with the exception of objective 2, which was only partly met. This was because that objective relied heavily on being able to undertake in vivo experimental work with the viruses, which was not possible after July 1997, when experimentation stopped because of the refurbishment of the ozone disinfection unit. Likewise some targets within other objectives were not met or delayed for the same reason, until alternative arrangements for experimentation were made.

1. SVCV/PFRV studies

SVC is a notifiable disease that affects carp and other species, mainly cyprinids, and the causative virus is serologically related to the non-notifiable PFR, which affects mainly pike, cyprinids (but not carp under natural conditions) and other species. During investigations into cyprinid mortalities, viruses have been isolated at the laboratory that, on the basis of serological tests, were serologically 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 try to determine their significance for carp, and to try to determine their relationship to SVCV, so as to provide advice as to whether they should be controlled.

Initial studies focussed on developing an in vivo challenge system for the viruses. The viruses were grown in cell cultures, titrated, and known amounts given by intraperitoneal (i/p) injection into test species, or added to the water in which the fish were swimming (bath infection). The studies showed that viruses that were of low virulence by the bath method had greater virulence by injection, a common finding with fish viruses. Injection of virus and co-habiting the fish with non-injected fish was another suitable method for assessing the virulence of the viruses. Subsequently comparisons were made of the virulence of certain of the intermediate viruses, PFR and SVCV for cyprinid hosts. The results are shown in the Table 1. Three of the intermediate viruses were not pathogenic for carp by bath infection, but were of low virulence by i/p injection. SVCV isolates were moderate to highly pathogenic to carp under the same conditions. An intermediate isolate from roach had low pathogenicity for roach and carp, but had higher pathogenicity for orfe, whereas an SVCV isolate had similar pathogenicity for roach but was not pathogenic for orfe. This suggests that there is a degree of host specificity amongst the virus isolates, and that the intermediate isolates do pose a threat to certain cyprinids.

CSG 15 (1/00) 4 Project Research on viruses of fish and shellfish MAFF title project code FC1105

Table 1. Pathogenicity of 10 rhabdovirus isolates for cyprinids.

Cumulative mortality (%) at day 30 post infection (Virus challenge – intraperitoneal injection and/or bath) Isolate Original Carp Carp Roach Tench Orfe host (i/p) (i/p) (bath) (i/p) (i/p) PFR: S 64 Tench 21 -* - 0 - PFR: GRV Grass carp 14 - - - - Intermediate: Roach 16 0 24 12 56 80560 Intermediate: Orfe 17 0 0 4 7 880137 Intermediate: Tench 7 0 - 0 - 950237 Intermediate: Carp 13 - - - - 970469** SVCV: 880062 Carp 88 20 20 12 0 SVCV: M2-78 - 60 - - - SVCV: 940500 Goldfish - 72 - - - SVCV: M 27 Carp 84 46 - - -

*- = not done **This isolate was later shown to be genetically similar to SVCV isolates from China

Later on in the lifetime of the project PFR-like/intermediate isolates were isolated during diagnostic tests (under contract FA001) on moribund or dead tench from a lake in England, from from the same lake, and from moribund or dead bream and roach from other sites in England. PFR-like/intermediate virus isolates from bream mortalities in Northern Ireland were also received from colleagues in Northern Ireland. Some of these isolates were used in virus transmission trials. One virus isolate from bream was virulent for bream, but another isolate from bream and an isolate from tench were not. The bream that were affected by the bream isolate showed many of the clinical signs of SVC disease. None of the isolates were virulent for carp, but neither was a control SVCV isolate. Those carp had been kept at the laboratory for some months and may not have been as stressed as carp under natural conditions i.e. the carp had been fed well, were not overcrowded, not suffering from other disease, were not immunocompromised – and so were resistant to the virus. The effect of stress on virus-challenged fish should be determined in future experiments.

Further serological studies were done on a range of UK isolates and other “PFR” or SVCV isolates obtained from colleagues abroad. The isolates were compared`in immunoassays or virus neutralisation tests using antisera raised against both SVCV and PFRV. Many isolates could always be identified as PFRV, SVCV or intermediate by both methods, but for some other isolates the distinction between SVCV and intermediate was difficult to make. This was possibly a reflection of the fact that in the two serological tests the reactions of the antisera with different viral epitopes were being measured, and/or because of the relatively low titres of the antisera. It is notoriously difficult to produce high titre antisera against these viruses, and in trying to produce high titres, there is a possibility of losing specificity. The antisera are specific enough to distinguish SVCV from PFR, but possibly not specific enough to distinguish between intermediates and SVCV. Interestingly, certain

5 Project Research on viruses of fish and shellfish MAFF title project code FC1105 isolates identified at other laboratories as being PFR were found to be intermediates in this study. The isolates from tench, bream, crucian carp and roach described above were identified as intermediate isolates.

Later studies done in conjunction with seedcorn project DP105 enabled nucleotide sequence comparisons of a region of the glycoprotein gene of the viruses to be made. Primers for use in the reverse-transcription polymerase chain reaction (RT-PCR) were designed using published sequence data for SVCV, and modified to enable PFR and intermediate isolated to be amplified and sequenced (under DP105). Those studies enabled more precise comparisons of the virus isolates to be made, and phylogenetic analysis of 38 virus isolates identified four genogroups, I – IV. Genogroup I comprises isolates which we regard as SVCV, and has Rhabdovirus carpio as the type species. Within this genogroup there are distinct sub-groupings, and one such sub-grouping comprises SVCV isolates from cold-water ornamental fish imported from China (isolated during investigations under FA001). Genogroup II, comprises only one isolate, from grass carp. Genogroup III comprises two isolates, the original PFR (the type species) and one other isolate from pike. Genogroup IV comprises the English and Northern Ireland bream, tench, roach and crucian carp isolates, and other isolates from the UK and Europe previously designated as PFR, or as intermediates; that group has a tench virus as its type species.

Although there is a serological relationship between isolates in the four genogroups, the nucleotide sequence differences between the viruses allocated to genogroups II, III and IV are such that cannot be regarded as SVCV on a genetic basis. Genogroups II, III and IV each shared less than 60% nucleotide sequence similarity with Genogroup I. However, laboratory studies (ours and of other scientists) and field observations show that the viruses are strongly associated with, or cause SVC-like disease in a range of cyprinid species. We are recommending to the scientific community that “spring viraemia of carp” is no longer a relevant name for the disease caused by these viruses but that “spring viraemia of cyprinids” is more appropriate. Changes to the legislation will need to be considered.

2. VHSV studies

Immediately prior to the commencement of the project, VHSV was isolated in 1996 at this laboratory (under the predecessor of this project) from herring from Rye Bay (English Channel). The fish had been collected under project AE003 in which there is an annual fish disease monitoring survey in coastal waters of England and Wales and parts of the North Sea. Similar surveys were also done in 1997 and 1998, and approximately 500 fish were tested in 1997 and 700 fish were tested in 1998. However, VHSV was not isolated in either year, despite the use of a wider range of susceptible cell cultures and the use of methods to enhance the uptake of viruses into the cells.

When VHSV was isolated from herring it was isolated in Epithelioma papulosum carpio (EPC) cells, but not in bluegill fry (BF-2) cells. That was surprising as BF-2 cells are regarded as being one of the cell lines of choice for isolating the virus, at least from freshwater fish. Indeed, a study at this laboratory, started under the predecessor to this project and continued under this contract, on the susceptibility of 12 cell lines to 13 VHSV isolates from marine and freshwater fish from North America and Europe showed that all of the isolates grew well in BF-2 cells. The viruses were inoculated onto the cells at the same concentration and observed for the appearance of destruction of the cell monolayer (cytopathic effect, CPE) for a seven-day period. If there were less than 25% cells showing CPE, the culture supernatant was passaged at a 1:10 dilution onto fresh cell monolayers. The approximate proportion of cells showing CPE at 7 days post inoculation before and after passage (if appropriate) are shown in Table 2.

One of the disadvantages of this type of study is that the virus may become adapted to the cell line in which it is grown, which may influence it’s ability to grow in a different cell line. Ideally, the viruses should be used to infect fish, and this type of study done on the viruses recovered from the fish. However it was not possible to infect the original hosts of all of the viruses studied and in order to lessen the “adaptation” effect, the viruses had had, as far as possible, less than six passages in cell culture from isolation from fish. It can be seen from the results that all the isolates passage readily into at least one cell line other than the one previously used for their culture, suggesting that there is no “adaptation” effect. The Atlantic salmon cell line, AS-6, is the only one of those tested that is completely refractory to all the isolates; interestingly, the isolates grew well in other cell

6 Project Research on viruses of fish and shellfish MAFF title project code FC1105 lines derived from Atlantic salmon. Very few isolates grew well in the TF cell line (from turbot) before passage, except for two isolates originally isolated from turbot.

Table 2. Relative sensitivity of 12 fish cell lines to VHSV isolates

Cell line VHSV isolate European Freshwater European marine North American (Pacific coast) VHSV F1 rainbow trout IW rainbow trout V16 rainbow trout 1458 rainbow trout Cod rhabdo Atlantic cod H17/5 Atlantic cod 814/94 turbot 7321 turbot AK 93 Pacific herring BC 93 Pacific herring NA-1 Coho salmon NA-2 Chinook salmon NA-6 Pacific cod

BF-2 **** **** **** **** *** **** **** **** **** **** **** **** **** RTG-2 */**** **** **** **** 0/** */**** ** **** **** ** 0/**** **** ** EPC **** **** **** **** */* **** **** 0/* **** **** **** **** **** FHM **** **** **** **** *** **** ** **** **** **** **** **** **** AS **/**** ** *** ** **** **** **** **** **** **** **** **** **** ASE **** **** **** **** *** **** **** */**** **** **** **** **** **** AS-6 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 BB ** 0/** ** ** 0/* **** ** 0/** **** **** *** *** *** CHSE ** ** ** **** 0/**** ** 0* */**** ** **** **** **** **** -214 RTE *** *** **** **** ** ** *** **** **** ** *** **** *** SBL * *** **** **** *** **** **** 0/** **** **** **** **** ** TF */*** 0/**** *** 0/* 0/* 0/**** **** **** ** ** 0/*** ** 0/***

0 = no growth, * = up to 25% CPE, ** = 25-50% CPE, *** = 50-75% CPE, **** = 75-100% CPE, / = passage (CPE before passage/CPE after passage), _____ = previous cell line for culture of virus

AS = Atlantic salmon ASE = Atlantic salmon embryo AS-6 = Atlantic salmon 6 BB = brown bullhead CHSE-214 = chinook salmon embryo 214 FHM = fathead minnow RTE = rainbow trout embryo RTG-2 = rainbow trout gonad 2 SBL = sea bass larvae TF = turbot fin

In 1997, BF-2 and EPC cells were again used for monitoring fish for VHSV and although no VHSV was isolated, RT-PCR studies (DP105) showed that the VHSV genome could be detected in the fish. As a result of that finding, in 1997 the fish samples were also inoculated onto FHM and CHSE-214 cells, with and without pre-treatment of the cells with polyethylene glycol (PEG) 20000. PEG has been shown to enhance the uptake of many viruses into cells. Again no VHSV was isolated, but the virus genome was detected by RT-PCR. The virus genome appeared subjectively to be present in large amounts in some fish. The reason why the virus was not isolated in the cell lines that studies have shown will replicate the virus to a high level is unknown, but may be because some marine isolates require different conditions for attachment to cells or replication in the cells. The herring isolate, which did not initially grow in BF-2 cells, was readily passed onto them from the

7 Project Research on viruses of fish and shellfish MAFF title project code FC1105

EPC cells. This suggests that having replicated in the EPC cells, the virus was readily adapted to growth in cell culture and could then be passaged onto other cell lines. This also demonstrates the importance of using wild-type virus in fish extracts for cell-line susceptibility studies.

It has only been in recent years that VHSV has been isolated from the marine environment, and there is a lack of information on such isolates. One important property of the viruses when risk assessments are being undertaken is how long the virus will persist in the marine environment. However, a search of the literature showed that only a short-term (1 hour exposure) study or a study incorporating a serum supplement (which protects the virus) had been done. A study of the survival of three North American marine isolates (VHSV Genogroup I), three European marine isolates (VHSV Genogroups II and III) and three European freshwater isolates (VHSV Genogroup III) in seawater at 4, 10 15 and 20°C was undertaken. The viruses were either diluted in seawater or cell culture maintenance medium and maintained at the different temperatures. The viruses were titrated immediately (time 0), then at days 1, 2, 3, 7, 14, 21, 28 and 35. The survival time varied between strains, but it could be for up to 4-5 weeks with an inverse correlation between survival and water temperature (Table 3). That survival time is a serious risk factor in the transmission of the virus.

Table 3. Stability of nine VHSV isolates in seawater

Survival time (days) Water temperature (°C) Isolate Host/region 4 10 15 20 NA-1 Coho salmon 14-21* 3-7 3-7 3-7 North America NA-6 Pacific cod 7-14 3-7 3-7 3-7 North America AK-93 Pacific herring 7-14 3-7 3-7 3-7 North America 83:53 Rainbow trout 7-14 7-14 3-7 3-7 Denmark Spanish Rainbow trout 3-7 7-14 3-7 0-1 VHSV Spain 3592 Rainbow trout 28-35 3-7 3-7 3-7 Denmark 814/94 Turbot 7-14 3-7 3-7 3-7 Scotland 7321 Turbot 7-14 3-7 3-7 1-2 Germany Cod rhabdo Atlantic cod 14-21 14-21 14-21 1-2 Denmark

* Range is from day when virus was last detected to day when virus was no longer detected

NB All control isolates similarly diluted in medium were still viable after 35 days at all temperatures.

8 Project Research on viruses of fish and shellfish MAFF title project code FC1105

These results also revealed the importance of including several different isolates in such studies. The survival times of viruses under different environmental conditions in previously published studies have usually been based on results obtained with a single virus isolate. There is no reason to suppose that there would not be differences in survival times between different strains of those viruses, and so the results of those studies must now be treated with caution.

What little published information there was on the virulence of marine VHSV isolates suggested that they are not virulent for rainbow trout and certain other Pacific salmonids, but there were few data on their virulence for marine fish. Consequently virulence studies were undertaken using the Rye Bay herring, Gigha turbot and North American VHSV isolates. Most experiments followed the same format. Viruses were grown in cell cultures, titrated, and the fish were exposed to a known dose of virus (expressed as plaque-forming units [PFU]/ml) by a bath infection. Negative controls were always included, as were positive controls where possible.

The first experiment investigated the virulence for turbot of a VHSV isolate from UK waters, the turbot isolate from the island of Gigha, Scotland, (814/94), compared with the virulence of a German turbot isolate (7321) and an isolate from rainbow trout in freshwater from Denmark (3592). The results are shown in Table 4.

Table 4. Virulence of VHSV isolates for 7 cm turbot

Isolate Concentration in the water Cumulative mortality (%) at day 35 post infection (PFU/ml) 814/94 (turbot, Gigha) 105 70 814/94 104 57 814/94 103 47 7321 (turbot, Germany) 105 7 7321 104 10 7321 103 0 3592 (rainbow trout, 105 0 Denmark) 3592 104 10 3592 103 3

There was a marked dose-response effect for the Gigha turbot isolate, which was shown to be highly virulent for turbot. However, the German turbot isolate and the rainbow trout isolate produced only low-level mortalities. The former virus was originally associated with mortalities in turbot, but may have lost virulence during passage in cell culture. The rainbow trout isolate was still virulent for rainbow trout, and so a host specificity of the isolates has been demonstrated. Subsequently 20 cm turbot were infected with the Gigha turbot isolate at either 104 or 103 PFU/ml, which produced cumulative mortalities of 27% and 20% respectively by day 42 post infection. This indicates a decrease in the susceptibility of turbot with age/size.

We were unable to test the virulence of the Rye Bay herring isolate in herring, but were able to test it in 7 cm turbot. An infection with 105 PFU/ml produced 37% mortality by day 35 post infection. The mortality was lower than that produced by the Gigha turbot isolate at the same concentration, which again suggests a degree of host specificity of the isolates, but also indicates that marine fish are still at risk from VHSV isolates from other marine fish.

The susceptibility of 4-8 g rainbow trout to a bath infection with the Gigha turbot isolate or Rye Bay herring isolate was tested in comparison with a Danish rainbow trout isolate (3592). The latter isolate produced 35%

9 Project Research on viruses of fish and shellfish MAFF title project code FC1105 mortality by day 35 post infection, but the two marine isolates were completely avirulent for the fish, which all survived the challenge. These observations support the findings of other workers using different VHSV isolates that isolates from the marine environment are avirulent or have only low virulence for rainbow trout.

As salmon may be exposed to marine VHSV isolates, their susceptibility was tested both in freshwater and in seawater. Bath infections of 20 g salmon were done with virus concentrations of 104 PFU/ml. The Gigha turbot isolate was avirulent for the salmon, but 18% of the salmon infected with the Rye Bay herring isolate died (on one day). Virus was not re-isolated from those fish, and so the mortalities cannot be confirmed to be specific to the virus. However, 32% of salmon infected with rainbow trout isolate 3592 died by day 24 post infection. The fish exhibited severe haemorrhaging of the dorsal musculature, and virus was re-isolated from them. This is a significant result, as Atlantic salmon in freshwater have not been reported as being susceptible to VHSV by bath infection, despite many previous infection trials at other laboratories. The 3592 virus re- isolated from the salmon was used to infect more salmon from the same stock, now four months older than used in the initial experiment. The mortality was lower than in that experiment, 20% at day 32. However the fish were stressed at day 39 post infection, and from day 44 until the end of the experiment at day 49, there were mortalities each day, reaching a cumulative total of 32% by day 49 post infection. The experiment showed that the virulence of the isolate had not increased by passage through the salmon, but the results may have been obscured by the age difference in the two groups of fish.

A larger scale experiment was undertaken to assess the virulence of several VHSV isolates from the marine environment for salmon in seawater. However, the experiment was made difficult to interpret because there were mortalities in control fish, but there were mortalities above that level in some fish groups infected with some of the virus isolates. As stress often appears to affect the level of mortalities caused by some viruses, the fish were stressed by reducing the water flow for four hours on day 39 post infection. The results are shown in Table 5, and have been expressed as relative % mortality (RPM) in order to compensate for the control mortalities.

Table 5. Virulence of VHSV isolates for salmon in seawater

Isolate RPM* to day 39 RPM from day 40 to (stress) post day 49 post challenge challenge NA-1, coho salmon North 11.4 14 America AK-93, Pacific herring 11.4 41.9 North America 814/94, turbot 9.1 11.6 Gigha H 17/5, Atlantic cod 0 2.3 (waters off Scotland) 96-43, Atlantic herring 0 100 Rye Bay 3592 rainbow trout 15.9 16.3 Denmark

*Relative % mortality, = (1-(% survival infected fish / % survival control fish)) x 100

The RPM of the fish infected with the isolate from coho salmon was 11.4%, and that figure did not change significantly after the fish were stressed. Similar results were recorded with the isolates from turbot and

10 Project Research on viruses of fish and shellfish MAFF title project code FC1105 rainbow trout. In contrast, the RPM of the fish infected with the Pacific herring isolate increased from 11.4% before the salmon were stressed to 41.9% post stress. The Atlantic herring isolate and the Atlantic cod isolate were both avirulent for the salmon before and after stress. These results indicate that certain marine VHSV isolates may be virulent for salmon in seawater, and that stress may affect the outcome of the infection. Subsequent to this experimental infection, VHSV was reported to have been isolated from Atlantic salmon in the marine environment in Canada; the source of the virus was thought to have been a marine fish.

Overall these results present a complex picture for disease management purposes. VHSV isolates of marine origin do not appear to pose a significant threat to commercial fish species in freshwater, although the effect of factors such as stress or immunosuppression on the susceptibility of those species to the marine isolates has yet to be tested. Certain of the marine isolates do, however, pose a threat to commercial species in seawater. These virulence experiments need to be repeated/continued in order to have a clearer picture of the significance of the for freshwater fish.

Further studies on the virulence of VHSV isolates for fish had to be abandoned because of the refurbishment of the ozone disinfection unit at the laboratory.

For risk assessment purposes it is important to know whether a new (marine) VHSV isolate may be virulent for freshwater or marine fish. Experiments were initiated to determine whether it was possible to predict the in vivo virulence of a virus from an in vitro property of a virus. Determining what to measure was problematical, but because virulence/infectivity may be related to the ability of a virus to attach to and penetrate into a cell, attachment of viruses to cell cultures was investigated. The glycoprotein spikes on the VHSV virion attach to cell membrane phospholipids, and the extent of phospholipid binding is pH dependent. One consequence of virus-phospholipid binding is cell to cell fusion, and it has been shown in the literature that there is a pH optimum for that fusion using a virulent VHSV isolate from rainbow trout. Preliminary experiments were started to determine whether there were differences in the pH optima of several different VHSV isolates and if so, whether there was any correlation between the pH optimum and factors such as origin of the virus, or its virulence for any species. Replicate EPC and BF-2 cell cultures were infected with VHSV isolates at a multiplicity of infection of 3 (i.e. 3 virus particles/cell) and incubated for 16 h. The medium was then removed from the cells and replaced with medium buffered to pH 4.0, 4.5, 5.0, 5.5, 6.0, or 7.0 for 1 h after which the cells were fixed and stained. The proportion of fused cells to non-fused cells (measured by counting nuclei) in five randomly selected fields were calculated at each pH step, and the pH optima for the different viruses calculated (Table 6).

Table 6. pH optima for fusion of EPC and BF-2 cells caused by VHSV isolates

pH Optimum Cell line Isolate EPC BF-2 3592 rainbow trout 5.5 5.5 Denmark 07-71 rainbow trout 4.5-5.5 4.5-5.5 France 814/94, turbot 5.0 4.0-5.0 Gigha 96-43, Atlantic herring Not done 4.0-5.0 Rye Bay

These data are preliminary, but they do suggest a trend, and the results from both cell lines are similar. VHSV isolates 3592 and 07-71 are virulent for rainbow trout and have pH optima as 5.5 for the former and 4.5-5.5 for

11 Project Research on viruses of fish and shellfish MAFF title project code FC1105 the latter, whereas 814/94 and 96-43 are avirulent for rainbow trout and have lower pH optima of 4.0-5.0. These results must be repeated before firm conclusions can be made; unfortunately, other priorities prevented further work on this during the project. Interestingly, subsequent to this work being completed, a report was published which showed that an avirulent variant of VHSV isolate 07-71 had a lower pH optimum compared with the wild type virus. These results relate to the virulence of the VHSV isolates for rainbow trout, but so far the data are too few to be able to make predictions concerning the virulence of these isolates for marine species. This work should be continued to determine whether the pH optimum for fusion really is a predictor of virulence of VHSV isolates.

3. Production and assessment of monoclonal antibodies (MAbs) against VHSV, IHNV and SVCV and preparation of antisera against other viruses as necessary.

Production of MAbs against VHSV and SVCV was started under the predecessor to this project, and that production and testing was continued. However, despite testing nearly 100 clones for each virus, no specific VHSV or SVCV secreting clones were identified. Further work on production of MAbs against VHSV, IHNV and SVCV started but could not be continued because of staff shortages. An IHNV MAb was obtained from colleagues in Denmark, and it was used successfully in a standardised immunoassay for that virus. Improvements were made to the standardised immunoassay used to detect SVCV and VHSV, as a result of greater experience with their use. The SVCV assay is used as a preliminary screen of carp suspected of having clinical SVC. Initial tests used material derived from experimental infections of fish but further refinements were needed as a result of experience with field samples. Immunoassays for the direct detection of VHSV, IHNV and SVCV are available for use in diagnostic tests under FA001 and the performance of the assays have been kept under review; the assay for SVCV has been most widely used in FA001. All three assays have been amplified by use of an avidin/biotin system to increase their sensitivity, and following further standardisation, are now used as appropriate to identify the cause of cytopathic effects in cell cultures, both in this project and in FA001. The VHSV assay was used in retrospective tests on herring tissues, some of which contained VHSV, and false positive reactions were observed as a result of high background reactions. The test was subsequently modified and more stringent assay conditions applied to overcome that problem. A polyclonal antiserum against SVCV has been produced for use in the rapid tests.

Birnavirus (IPNV-like) isolates from fish from several Southeast Asian countries were sent to this laboratory, as the OIE Reference Laboratory for IPNV, from colleagues in Singapore for confirmation of identification. Initial testing showed that they did not belong to the two established birnavirus serogroups, and an antiserum was produced against one of the viruses. That confirmed that the viruses were representatives of a new birnavirus serogroup. Subsequently more birnaviruses were isolated from fish imported into Singapore and were sent to this laboratory for identification. They too appeared to be different from the established birnavirus serogroups and from the newly recognised third serogroup. Antisera have been produced against two of those isolates, and it does appear that they may represent a fourth serogroup. As ornamental fish are imported into this country and tested at the laboratory for viruses under FA001, these antisera will be of value in identifying any viruses isolated during the diagnostic tests. The molecular masses of the two double-stranded (ds) RNA segments of the viruses were qualitatively compared after extraction of the RNA and electrophoresis on 6% polyacrylamide gels. Differences in the migration of the two RNA segments were seen between isolates representing the third and the putative fourth serogroup, and between those two serogroups members of the original two serogroups. Electrophoresis of the extracted nucleic acid was a quick way to confirm that the viruses actually were birnaviruses in the initial absence of specific neutralising antibody. Only after a larger number of isolates are tested will there be sufficient data to determine whether electrophoresis of the extracted nucleic acid can also be used to predict the serogroup to which a birnavirus isolate may belong. That information will aid in specifically targeting subsequent neutralisation tests to confirm the serological identity of an isolate.

4. Identification of novel viruses isolated during diagnostic investigations

An aquareovirus was isolated from brown trout during routine monitoring under FA001. The pathogenicity of that virus for rainbow trout was tested as brown trout were not available. The virus was inoculated into the fish

12 Project Research on viruses of fish and shellfish MAFF title project code FC1105 by the i/p route and they were then observed for 33 days. There were no mortalities, and no gross external signs were observed. However, titration of virus from internal organs showed that the virus had replicated in the fish, and histological examination revealed haemorrhages and focal necrosis in the liver. Whilst the significance of this virus for brown trout is unknown, these results show that it can be transmitted to rainbow trout and the effect of the virus on the liver could possibly make the fish more susceptible to other pathogens or depress their growth rate.

During the marine fish surveys for VHSV, viruses other than VHSV were isolated. In 1997 birnavirus serotype B1 was isolated from Atlantic herring from Liverpool Bay and from Atlantic cod in Rye Bay, and birnavirus serotype A2 was isolated from Atlantic cod in Camarthen Bay. Birnaviruses are ubiquitous in both the marine and freshwater environments, and it is no surprise that they were isolated during the surveys, although the herring represents a new host for the virus. However, it was curious that the viruses were all isolated in the same year, and birnaviruses were not isolated in 1996 or 1998. One of the isolations was from Rye Bay, from where VHSV was isolated the previous year, but there may be no significance in that. In 1998 an aquareovirus was isolated from haddock from Off Amble, again a new host record for that virus. The virus was only cultured in cells that had been pre-treated with PEG; such pre-treatment of cells is recommended for future surveys. Aquareoviruses have 11 segments of ds RNA, which can be separated by electrophoresis into three broad size classes, according to the molecular mass of the RNA segments. The pattern of migration of the RNA segments has been used to group the viruses (as electropherotypes), and their genetic relatedness has been determined by RNA-RNA hybridisation. The RNA migration patterns of the haddock and brown trout aquareoviruses were compared on 6% polyacrylamide gels after extraction of the RNA, and although distinguishable from each other, both viruses had a similar migration pattern and should be allocated to the same electropherotype. Whether the viruses are genetically related can only be resolved by hybridisation studies. The significance of the birnaviruses and aquareovirus for their hosts is unknown. Some aquareoviruses can cause high mortalities in their hosts, but the majority appear to have little direct impact. Likewise some birnaviruses are highly pathogenic for their hosts, whilst others are not.

An agent causing a syncytial CPE in EPC cells has been observed on many occasions during routine monitoring tests on carp, and during specific diagnostic tests on moribund carp under FA001. The agent is usually slow growing, but the growth rate can vary considerably. It can take many weeks for the agent to grow to a sufficient level for undertaking specific tests and that has meant that studies on the agent have been protracted and have often been deferred to undertake work of higher importance; the growth rate does, however, increase on passage into fresh cell cultures. At least two other laboratories (in Belgium and Germany) have also reported observing this agent during diagnostic tests using EPC cells. The CPE is reminiscent of that produced by aquareoviruses, but no such virus has been isolated, and ds RNA has not been observed after electrophoresis of extracted nucleic acid. The agent is sensitive to lipid solvents, but the determination of nucleic acid type has produced inconsistent results. Filtration studies have suggested that it may be larger than any known virus, and it may not even be a virus at all. Electron microscope (EM) studies have been done on thin-sectioned cell cultures infected with the agent, and marked changes and abnormalities have been seen in the cells. Noticeable amongst these has been the appearance of membrane- bound bodies, some of which appeared virus-like, and some of which could have been a small non-viral organism. Attempts were made to purify the virus-like material on sucrose density gradients and observe it under the EM after negative staining, but no convincing virus-like particles were seen. Infectivity trials using carp were done, but with inconsistent results. There were low to moderate mortalities in some trials, but no mortalities in others. In one trial groups of thirty carp were inoculated i/p with the agent isolated from six different diagnostic investigations, and observed for mortality for 30 days (Table 7)

Table 7. Pathogenicity of a cytopathic agent from EPC cells for carp.

Isolate Cumulative mortality (%) day 30 post infection 960451 3.3 960545 23.3 960625 16.6

13 Project Research on viruses of fish and shellfish MAFF title project code FC1105

960669 30 960885 10 960960 43.3 Control 3.3

The agent was isolated from dead fish infected with isolate 960960 and this was inoculated into further carp from the same group. However, there were no mortalities in the inoculated groups and the agent was not re- isolated.

In other trials, attempts were made to re-isolate the agent when mortalities did occur, but that was only successful in a small number of cases. This agent may not be a primary pathogen, and indeed may not be a pathogen at all. However, because it is cultured during diagnostic or monitoring tests it could interfere with the isolation of a true pathogen. The identity of this agent still needs resolving, and an antiserum should be raised against it so that it can be neutralised in fish tissue homogenates before they are inoculated onto cell cultures during diagnostic/monitoring tests.

5. Familiarisation with the isolation/identification of exotic viruses.

Koi carp herpesvirus

Unexplained mortalities in koi carp were reported from retail sites in this country and elsewhere receiving fish from Israel and Japan. No viruses were isolated at this laboratory when diagnostic tests were done on the fish under FA001, and histological examination did not show any pathology suggestive of a viral aetiology for the disease. However in the USA, virus-like particles were seen under the EM in similarly affected koi carp and a herpesvirus was isolated in a newly established cell line from koi carp, KF-1 cells. The virus did not grow in EPC or FHM cells at those first isolation attempts, and the KF-1 cells are the only cells as yet known to support the replication of the virus. Samples of the KF-1 cells and virus were obtained from the USA, and the virus was cultured in those cells at the laboratory. Unfortunately, the cells succumbed to a low-level bacterial contaminant, and all died. Some more cells have recently been received from the USA, and work on the virus will continue. In the meantime koi carp primary cell cultures (KFp) were initiated at this laboratory under FA001, and these supported the replication of the herpesvirus. Attempts were made to isolate viruses in the primary cells from stored extracts of tissues from koi carp mortalities submitted to this laboratory, but the cells were not sufficiently robust to survive inoculation with the extracts. However the cells are still being cultured and passaged, and may become more robust with time.

Transmission trials have been done with the herpesvirus. Ten carp were inoculated by the i/p route with virus cultured in KF-1 cells. On the 6th day post-infection two fish died and two were moribund, and were taken for histological examination. By the 9th day post infection all fish were either dead (6 fish) or were moribund and removed from the tank for further analysis. One control fish died on day 10. Histological examination of moribund fish revealed typical herpesvirus pathology in kidney, spleen and liver tissue. A filtered tissue extract from dead fish from the first trial was injected into 10 further carp. By the 6th day post-infection eight fish had died, and on the 7th day the remaining two fish were moribund, and were removed for further analysis. No control fish had died by day 10 post-infection. This virus is clearly very virulent for carp by i/p inoculation. Further trials are in progress to determine the virulence for carp by bath infection. If that virus caused the mortalities in the koi carp in this country it could cause high mortalities in native carp populations if conditions allowed transmission between the fish. Although koi carp are ornamental varieties and would not normally come into direct contact with native carp, the virus could be transferred by infected water, or by other fish that may have been in direct or indirect contact with infected koi carp.

Infectious salmon anaemia virus

The salmon head kidney (SHK-1) cell line in which the infectious salmon anaemia virus (ISAV) grows, ISAV and homologous antiserum were obtained from colleagues in Norway. The isolated virus was cultured in the

14 Project Research on viruses of fish and shellfish MAFF title project code FC1105 cells, and salmon extracts were inoculated onto the cells in order to become familiar with their handling characteristics, prior to their use for diagnostic purposes. Haemadsorption tests were done using salmon erythrocytes. A 0.05% suspension of salmon erythrocytes was added to control and infected cell cultures, which were then observed at intervals up to 1h. After 5 min the majority of the salmon erythrocytes had adsorbed to the infected cells, but none had adsorbed to the control cells. After 1 h the excess erythrocytes were removed and the cells were washed with buffer. All the erythrocytes were removed from the control cell cultures, but erythrocytes remained on the infected cell cultures. This is a rapid way of determining whether cells may be infected with the virus, in the absence of any CPE.

Infected cell cultures were fixed and sectioned for the EM, and virus was observed. Images have been lodged with the laboratory’s database for future reference. During the course of the project ISAV was isolated from diseased Atlantic salmon in Canada using the CHSE-214 cell line. Although it had been reported that the Norwegian ISAV did not grow in CHSE-214 cell, that work was repeated in our laboratory, as another publication had shown that there were vast differences in the susceptibility of CHSE-214 cell lines maintained at different laboratories. However, CHSE-214 cells at our laboratory also failed to support the replication of the Norwegian ISAV. It appears from other work that the failure of the Norwegian ISAV to grow in CHSE-214 cells is a property of the virus rather than of the cell line. Primers for use in RT-PCR were designed, under DP105, from published sequence data, and these were tested on cell grown virus and found to be suitable for identification of the virus.

Iridoviruses

An iridovirus from the European catfish was obtained from a colleague in Italy, and cultured in BF-2 cells. Although the catfish is presently not a species of interest to us, an iridovirus has been observed in sturgeon in Europe that are being imported into this country. Infected cell cultures were fixed and sectioned for the EM, and virus was observed. Images have been lodged with the laboratory’s database for future reference. It was the intention to develop a PCR identification method for iridoviruses using published data, but that was deferred because of other priorities.

An iridovirus isolated from carp with gill necrosis had previously been obtained from a colleague in Russia, but the virus had not grown in any of the cell cultures at the laboratory. However, it has been successfully cultured in the koi carp KFp cell line initiated at this laboratory. If that cell line does become more robust it will be a valuable aid when investigating carp mortalities.

Nervous necrosis virus

Nodaviruses or nodavirus-like agents have been reported to cause disease in Atlantic salmon and Atlantic halibut reared in Norway, and in European sea bass reared in several locations in the Mediterranean, and on the Atlantic coast of France. Infection with the causative viruses, termed “nervous necrosis viruses” (NNV), results in high mortalities in those species, and NNV also seriously affects the culture of many different marine fish species world-wide. The potential for the virus to be transmitted to freshwater species by fish returning from the marine environment is unknown, but must be kept under review. The striped snakehead (SSN-1) cell line (one of only three cell lines currently known to support the replication of nodaviruses) and a European NNV isolate were received from colleagues in Stirling, and a purified preparation of a Japanese NNV were received from colleagues in Japan. Primers for use in RT-PCR were designed under DP105 from published sequence data, and these were tested on both European and Japanese virus and found to be suitable for identification of the viruses. A second cell line, from sea bass (SB), that supports the growth of NNV has been received from colleagues in Singapore. That cell line is being expanded at the laboratory, and will be used as an alternative/additional cell line for testing for NNV.

All the above work enhances the diagnostic capability of the laboratory, and enables us to respond rapidly should any disease outbreaks be caused by those viruses.

15 Project Research on viruses of fish and shellfish MAFF title project code FC1105

6. Use of the antibody screening test.

Three hundred and twenty cyprinid sera from 57 different sites were tested for antibody to SVCV by a competitive immunoassay, and fish from 21 sites were antibody-positive. SVCV was isolated from only one of those sites, and none of them had any history of SVC disease. The threshold for determining whether a serum was positive was that there should be >20% inhibition of the signal, and many of the fish that were positive had antibody levels producing 20 – 30% inhibition. However, all three sera tested from one SVCV- negative site had antibody levels producing 42, 76 and 77% inhibition, and checks indicated that they were not false-positives. The antibody levels in the sera from the SVCV-positive fish produced 23 and 28% inhibition, and two other fish from that site were antibody negative. Further analyses need to be done, and the threshold level may have to be adjusted upwards, if some of these results are false-positives. If they are not false positives, and the very high antibody levels are most likely not, the interpretation is that a) the fish have been exposed to the virus in the past, and have eliminated it b) it is present in the fish, but in tissues not tested for virus, c) it is present in the fish but at levels or in a form that does not grow in cell culture. Recent results from contract FC1113 have suggested that the latter may be the case. In fish surviving experimental infection with a virus having low virulence, virus was found mainly in the pancreas by in situ hybridisation, and that tissue is not taken for virus isolation. These results may lead to a re-evaluation of the method(s) used for detecting SVCV carrier fish.

7. Assessment of new diagnostic tests.

A watching brief has been kept on developments in diagnostic techniques, but apart from minor modifications to existing procedures, there have been few significant advances published. Some of these have been reported upon above, such as the use of SHK-1 cells for culturing ISAV, and SSN-1 and other cells for culturing nodaviruses.

The PCR is a method that is likely to be used in diagnostic tests and that is being used and evaluated under DP105, FA001 and this project. Currently it has been used successfully in the surveys for VHSV in marine fish, to confirm the cause of mortalities in experimental infections and to provide further information on the genetic relationships between viruses. Further validation work is needed before it could be accepted as a monitoring or diagnostic tool for statutory purposes, and that work will be undertaken in a new project starting in April 2000.

Research is being undertaken at this laboratory under FC1113 on in situ hybridisation, which is a method whereby the specific nucleic acid component of viruses (or other pathogens) can be localised in sections of tissues from fish, or any other organism. This is a powerful tool that can be used as a stand-alone method, but it can also be used in conjunction with histopathological examination of the same tissues. The assessment of that method is being done as part of FC1113.

8. Maintenance of a knowledge base of virus diseases of fish and shellfish.

Our knowledge base of virus diseases of fish and shellfish viruses is constantly being kept up to date, so that we have ready access to information when advice is sought by MAFF, or from within CEFAS e.g. Chief Advisor or the Inspectorate. This has been done by:

a) conducting regular searches of the scientific literature and obtaining reprints of all articles on fish and shellfish viruses not in journals held by the library. All such accessions are entered into the library database.

b) corresponding with/telephoning colleagues at other laboratories and entering into a two-way interchange of information and biologicals (e.g. virus isolates, antisera, cell cultures).

16 Project Research on viruses of fish and shellfish MAFF title project code FC1105

c) attending scientific meetings/workshops etc and talking directly with colleagues from other laboratories. Officers working on this project were on the organising committee of the IV International Symposium on Viruses of Lower Vertebrates held at this Laboratory in 1998.

d) undertaking collaborative projects with colleagues from other laboratories.

Acknowledgements: This project has benefited greatly from the input and assistance of colleagues working on projects FA001, DP105, AE003 and FC1113, and that help is gratefully acknowledged here.

17 Project Research on viruses of fish and shellfish MAFF title project code FC1105

Appendix: Publications and other outputs:

Publications/Reports

Dixon, P.F. (1997). Viral diseases of carp and catfish. In R. Gudding, A. Lillehaug, P.J. Midtlyng, & F. Brown (Eds.), Fish Vaccinology. (pp. 221-232). Basel: S.Karger.

Dixon, P.F. (1999). VHSV came from the marine environment: clues from the literature, or just red herrings? Bulletin of the European Association of Fish Patholologists 19, 60-65.

Dixon, P.F. (compiler) (1999). Interim report on studies of rhabdoviruses isolated from cyprinid fish in 1999. Prepared for MAFF customer.

Dixon, P.F., Feist, S., Kehoe, E., Parry, L., Stone, D.M., & Way, K. (1997). Isolation of viral haemorrhagic septicaemia virus from Atlantic herring, Clupea harengus, from the English Channel. Diseases of Aquatic Organisms, 30, 81-89.

Dixon, P.F & de Groot, J. (1998). Detection of antibodies against infectious pancreatic necrosis virus in rainbow trout blood collected on filter paper discs. In Methodology in Fish Diseases Research (A.C. Barnes, G.A., Davidson, M.P. Hiney and D. McIntosh, Eds.), pp 241-246. Fisheries Research Services, Aberdeen.

Parry, L. & Dixon, P.F. (1997). Stability of nine viral haemorrhagic septicaemia virus (VHSV) isolates in seawater. Bulletin of the European Association of Fish Pathologists, 17, 31-36.

Stone, D.M., Way, K. & Dixon, P.F. (1997). Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia (VHS) viruses from different geographic areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.). Journal of General Virology 78, 1319-1326.

Way, K. (1998). Problems arising from the use of immunoassays for the detection of viral pathogens directly in fish tissues. In: Methodology in Fish Diseases Research (A.C. Barnes, G.A. Davidson, M.P. Hiney, and D. McIntosh, Eds.), pp. 195-201. Fisheries Research Services, Aberdeen.

Presentations at meetings

Avery, S., Chambers, E., Dixon, P.F., Feist, S., Mandhar, H., Parry, L., Stone, D.M., Strømmen, H.K., Thurlow, J.K., Lui C.T.Y. & Way, K. (1998). Four years of monitoring for fish viruses in marine waters around the UK. Poster presentation at IV International Symposium on Viruses of Lower Vertebrates, Weymouth. Abstract PP-15.

Chew-Lim, M., Ngoh, G.-H., Chong, S.Y., Chang, S.F., Kueh, S.L.F, Way, K. & Dixon, P.F. (1999). Birnaviruses of potential new serogroups isolated from tropical fish. Oral Presentation at the 4th International symposium on Diseases in Asian Aquaculture, Cebu City, Philippines. Abstract OP 45.

Dixon, P. F. (1997). Viral diseases of carp and catfish. Oral presentation at the International Symposium on Fish Vaccinology, Oslo.

Dixon, P.F. & Avery, S. (1997). Susceptibility of cultivated fish species to viral haemorrhagic septicaemia virus isolates from marine hosts. Oral presentation at VIII International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Edinburgh. Abstract O-83

18 Project Research on viruses of fish and shellfish MAFF title project code FC1105

Dixon, P.F. & Avery, S. (1997). Susceptibility of cultivated fish species to viral haemorrhagic septicaemia virus isolates from marine hosts. Poster presentation at International Symposium on Diseases in Marine Aquaculture, Japanese Society of Fish Pathology, Hiroshima. Abstract P-14, p 80.

Dixon, P.F & de Groot, J. (1997). Detection of antibodies against infectious pancreatic necrosis virus in rainbow trout blood collected on filter paper discs. Oral presentation at the Symposium on Methods in Fish Diseases Research, Aberdeen. Abstract p 52.

Rowley, H.M., Bryson, D.G., Campbell, S.J., Way,K. & Stone, D.M. (1999). Isolation of a rhabdovirus from wild bream. Poster presentation at IX International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Rhodes. Abstract P-i79.

Stone, D.M., Sheppard, A., Lui, C.T.Y., Taylor, G.R., Denham, K., Dixon, P.F. & Way, K. (1999). Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four distinct piscine vesiculovirus groups. Poster presentation at XI International Congress of Virology, Sydney. Abstract VP24.04, p 351.

Taylor, G., Forrest, A., Stone, D.M. & Way, K. (1998). Isolation of an aquareovirus from brown trout (Salmo trutta) in the United Kingdom. Poster presentation at IV International Symposium on Viruses of Lower Vertebrates, Weymouth. Abstract PP-02.

Way, K. (1996). Problems arising from the use of immunoassays for the detection of viral pathogens directly in fish tissues. Oral presentation at the Symposium on Methods in Fish Diseases Research, Aberdeen. Abstract p 50.

Way, K., Banyard, A.R., Dixon, P.F., Schmidt, N.T. & Stone, D.M. (1998). Is PFR virus simply SVC virus disguised in ‘pike clothing’?: comparative studies on rhabdoviruses from cyprinid fish. Poster presentation at IV International Symposium on Viruses of Lower Vertebrates, Weymouth. Abstract PP- 06.

Way, K., Dixon, P.F., Stone, D.M. & Schmidt, N.T. (1997). Comparative studies on rhabdoviruses isolated from cyprinid fish and their relationship to SVC virus and PFR virus. Oral presentation at VIII International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Edinburgh. Abstract O-41

Way, K., Stone, D.M., Dixon, P.F., Rowley, H.M. & Bryson, D.G. (1999). Isolation of rhabdoviruses from cyprinid fish species with a clinical disease indistinguishable from spring viraemia of carp: a proposal for the re-classification of rhabdoviruses causing haemorrhagic disease in cyprinid fish species. Oral presentation at IX International Conference “Diseases of Fish and Shellfish”. European Association of Fish Pathologists, Rhodes. Abstract O-096.

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