Section 3: Workshops Detection and typing of fish viruses

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1 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 158 Section 3: Workshops Detection and typing of fish viruses K. Einer-Jensen 1*, H. Björklund 2, S. Oreshkova 3, I. Shchelkunov 4, T. Vesely 5 and N. Lorenzen 1 1 Danish Veterinary Laboratory, Hangøvej 2, DK-8200 Aarhus N, Denmark. 2 Åbo Akademi University, Artillerigatan 6, FIN Turku, Finland. 3 State Research Center of Virology and Biotechnology, Koltsovo, Novosibirsk Region, Russia. 4 All-Russian Research Institute of Freshwater Fisheries, Rybnoe, Dmitrov Region, Moscow Province, Russia. 5 Veterinary Research Institute, Hudcova 70, CZ Brno Czech Republic Abstract The continuous appearance of new virus isolations from aquacultured fish makes it important to have efficient and reliable methods available, not only for identification of these viruses, but also for further typing in order to allow epidemiological analysis. Recent immunological and genetic assays for detection and typing of the rhabdoviruses infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV), spring viraemia of carp virus (SVCV) and European perch rhabdoviruses were presented and discussed during this workshop. Also, the need to standardise as well as validate these molecular techniques with traditional techniques as reference regarding sensitivity and specificity were discussed. Background Aquacultured fish represent an important industry in Europe, as well as in the neighbouring countries. Rapid diagnostic procedures for identification of viral pathogens are important in order to reduce losses in relation to outbreaks, and to prevent spread of the pathogens due to trade with live fish. In addition, methods suitable for epidemiological studies aiming at determination of the origin of infections are also required. As a result of the open trade with live fish within EU as well with neighbouring countries, the international standardisation of diagnostic procedures is important. Biotechnology-based assays including monoclonal antibodies, DNA probes, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) are being used for diagnostic in general in many fish disease laboratories, but often with different reagents. This makes standardisation complicated. In 1999 a collaborative EC-supported research project (EC FAIR CT ) entitled Diagnostic methods and reference panel of reagents for detection and typing of fish viruses was initiated, by 5 laboratories situated in Finland, Denmark, Czech Republic and Russia, respectively. With focus on fish rhabdoviruses, central objectives were to produce and characterise MAbs for identification and typing in immunoassays, probes and primers for diagnostic molecular assays as well as to sequence selected genes and analyse the phylogenetic relationship between a

2 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 159 number of rhabdoviruses. Finally, the idea was, based on the produced reagents, to develop prototype assays suitable for standardised diagnostic procedures. With the arranged workshop, we intended not only to present and discuss the obtained results, but also to bring focus on the advantages as well as the pitfalls related to the use of diagnostic molecular assays in comparison with the presently approved cell culture assays, which are used in the guidelines for survey programmes for detection of the fish rhabdoviruses (European Commission, 2000; Office International des Epizooties, 2000). These cell culture assays imply isolation of the viruses by inoculation of tissue homogenates or fluids onto susceptible cell lines, and are still considered to be the Gold Standard, although Lorenzen et al. (1999) reported a substantial variation in the ability of various laboratories to detect and correctly identify a set of four fish viruses sent to them from the European Reference Laboratory. Following isolation of virus in cell culture, various immunoassays such as immunofluorescence, ELISA and virus neutralisation are then used for identification of the isolated viruses (European Commission, 2000; Vestergaard Jørgensen, 1972; LaPatra et al, 1989; Office International des Epizooties, 2000). Similar immunoassays may be employed for direct detection of viruses in tissues from infected fish, but with a reduced sensitivity compared to the procedure including virus isolation in cell culture, and these assays are normally not appropriate as primary diagnostic assays on fish without clinical symptoms. Dr. Eva Maria Bernoth (Canberra, Australia) opened the workshop by discussing the use of sensitive molecular biology assays in primary diagnostics. In contrast to the traditional immunoassays, diagnostic assays based on amplification of pathogen specific gene sequences often have higher sensitivity than the cell culture isolation of virus. Whereas this basically appears as an advantage it also creates a number of specific questions, such as how to interpret and handle diagnostic cases where samples become positive in gene amplification assays such as PCR, but without any clinical signs of infection and no isolation of virus upon inoculation of cell cultures. Methods and assays presented during the workshop DNA hybridisation probes Several hybridisation assays using non-radioactive DNA probes have been developed for the confirmation of IHNV, VHSV and SVCV grown in cell cultures and for detection of selected virus isolates directly from tissues of infected fish (Batts et al, 1993; Deering et al, 1991; Gonzalez et al, 1997; Oreshkova et al, 1999). Actually, Batts et al. (1993) managed to develop a DNA probe assay, which can discriminate the genetically distant VHSV isolates from North America and Europe, respectively. These probes are now used in several laboratories in western North America to ensure that new isolates of VHSV from marine fish are of the North American genotype. Dr. I. Shchelkunov presented a study using the SVCV M and G gene specific biotin labelled probes of Oreshkova et al. 1999, on a panel of SVC viruses, as well as clinical samples from experimentally and naturally infected fish. These results were compared with traditional techniques of virus isolation and SVCV-serological survey. The probes ap-

3 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 160 peared to be quite robust when using cell culture, whereas a number of false positive/ negative reactions were observed when using fish tissue samples; the probe assay identified less positives among samples from fish in the acute phase of SVC, but more positives among fish in the carrier state when compared to cell culture assay. It was furthermore observed that the samples needed to be in the form of as much as possible purified target nucleic acid in order to avoid non-specific interactions of sample proteins and tissue nucleic acids with the probes. Optimisation of the procedure to minimize such unspecific interaction, as well as additional controls for specificity of the probes are therefore needed before it can be established whether such probes can be used in routine diagnostic work. Detection and typing using RT-PCR RT-PCR allows amplification of a few copies of a specified nucleic acid sequence from a pathogen into millions of copies in a short period of time, and a number of RT-PCR protocols using normal, semi-nested and nested PCR, as well as multiplex protocols have therefore also been developed for characterisation and detection of the fish viruses (Arakawa et al, 1990; Bruchhof et al, 1995; Einer-Jensen et al, 1995; Enzmann et al, 1998; Guillou et al, 1999; Miller et al, 1998, Strommen and Stone 1998, Williams et al, 1999; Yoshinaka et al, 1998). However, only a limited number of different virus isolates have been tested within these assays, and the question of general specificity for e.g. the primers in such RT- PCR assays remains to be addressed (Winton & Einer-Jensen, In press). Dr. Sven Bergman (Insel Riems, Germany) has initiated such work and he presented a study including a large representative panel of 55 VHSV and 12 IHNV isolates from Europe (freshwater and marine) as well as from North America. An internal RT-PCR control based on primers allowing amplification the b-actin mrna purified from fish tissue was also included in the test, in order to evaluate the efficacy of the method. The results revealed that the designed primers were virus specific (VHSV or IHNV) and recognized all isolates included in the panel. These promising results suggest that these primers may be included in a future RT-PCR method for virus identification, which can be used in parallel with the classical approved virological methods such as neutralisation test, ELISA and immunofluorescence. Efforts on making a RT-PCR protocol for identification of SVCV have not previously been described. However, Dr. S. Oreshkova and Dr. M. Koutna (Brno, Czech Republic) presented two different protocols developed on basis of the newly obtained sequence data presented at the workshop. N-gene products of Russian and European SVCV isolates were successfully tested in both protocols by normal, as well as nested RT-PCR, and the specificity of the amplified fragment was in one of the protocols additionally confirmed by digestion with the ClaI restriction enzyme of the product. Specific N-gene primers for discrimination of the two geno-subgroups (Russian and European SVCV isolates, respectively) identified during the above-mentioned sequence studies were also produced and the predicted genetically based differentiation was confirmed. Genotyping of VHSV isolates by RFLP Viral haemorrhagic septicaemia (VHS) virus has in the last decade been isolated from an increasing number of free-living marine fish species. These marine isolates are indistin-

4 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 161 guishable from fresh water isolates by traditional serological means, but they can be divided into a number of genotypes, which roughly correlates with the geographical site of isolation (Batts et al, 1993; Bernard et al, 1990; Bernard et al, 1992; Einer-Jensen et al, 1995; Snow et al, 1999; Oshima et al, 1993). The current genotyping methods involve labour intensive molecular techniques such as T1 nuclease fingerprinting, ribonuclease protection assay and sequence analyses (Kurath et al, 1995; Osima et al, 1993; Snow et al, 1999; Troyer et al, 2000). As an alternative to these methods, Dr. Katja Einer-Jensen presented a study on an alternative molecular typing assay based on enzymatic digestion of amplified G-gene products. The presented RFLP method was developed on basis of full length G gene sequence data from more than 60 geographically representative isolates. The developed method allows grouping of VHS virus isolates according to major geographical areas as well as detailed geno-subgrouping according to the performed phylogenetic analysis and appears to be suitable for standardised routine typing of new virus isolates with limited requirements of resources and equipment. Beyond this new typing assay, M. Snow (Aberdeen, Scotland, UK) stressed the need to understand the virulence characteristics of different genotypes, since such a molecular epizootiological tool is required for the management of marine viral haemorrhagic septicaemia virus (VHSV). Such a marker has not yet been identified in spite of the efforts of Betts & Stone (2000) were the entire genome of two virulent and two avirulent strains were sequenced and compared. Sequence analysis and phylogenetic studies Sequence comparison is definitely the most powerful method for genetical characterisation of virus isolates, but this method is still too costly to be suitable for daily routine typing of fish pathogens. Nevertheless, sequence analysis confirms the findings, revealing that genetic differences among VHSV and IHNV isolates appear to be most strongly related to the geographic location from which the isolate was obtained and not to year of isolation or host species (Basurco et al, 1995; Benmansour et al, 1994; Benmansour et al, 1997; Betts & Stone, 2000; Dixon et al, 1997; Emmenegger et al, 2000; Nichol et al, 1995; Stone et al, 1997; Snow et al, 1999). Limited sequence information of SVCV genes has so far been available. New sequence data of the whole viral G-gene, the P gene as well as a part of the N-gene for several Russian and European isolates where presented by Dr. Svetlana Oreshkova, Karina Kippasto (Åbo, Finland) and M. Koutna during the workshop. They found that the P gene was highly conserved and also that the G-gene was surprisingly well conserved among isolates of different origin compared to the G gene of VHSV and IHNV. Phylogenetic analysis of the sequence data of the selected SVCV isolates interestingly showed, that the isolates grouped in distinct clusters related to geographic location of isolation. In addition, the P gene analysis showed that three Moldavian SVCV isolates cluster closer to Vesiculoviruses than did the other isolates. As described in the following, these new sequence data have been used for the development of the first reported RT-PCR protocols for identification of SVCV.

5 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 162 Sequence data of G and L fragments of 13 poorly characterised perch rhabdoviruses were also presented by K. Kippasto in order to clarify their genetic relationship to other fish rhabdoviruses. The data reveal that these perch rhabdoviruses do not host a Nv gene, which is the characteristic of the fish Novirabdoviruses, they clade separately, and are closely related to the Vesiculoviruses. Typing of VHS virus isolates by monoclonal antibodies. Monoclonal antibodies are often used in routine diagnostic assays for identification of fish viruses in convenient immunoassays such as ELISA. Provided suitable antibodies can be supplied, such assays would also be suitable for a more detailed typing of new virus isolates with the aim of epidemiological analyses. Dr. Niels Lorenzen presented a study, which aimed at production of a panel of monoclonal antibodies that could be employed for a rapid typing of VHS virus isolates in a sandwich ELISA assay. Only antibodies with either strong or no reaction was included in the assay in order to ease interpretation of the results. Especially two MAbs (both anti N-protein) were of interest since they allowed typing of the 84 tested VHSV isolates into three groups: a) MAb1 recognized fresh water and none-baltic-marine isolates, b) MAb2 recognized Baltic-marine isolates, c) both MAb s recognized some old fresh water isolates and marine isolates, which had been isolated from VHS outbreaks. These findings suggest that a convenient ELISA assay for grouping of new virus isolates in relation to characteristics such as geographical origin can be established. Discussion Appropriate validation and quality assurance is required before approval of any of the described methods for identification or typing of the viral pathogens in routine diagnostics. As raised from the audience, a basic question is then to determine how an appropriate evaluation is to be performed, e.g. what number of isolates (and which) should be examined? What methods should be used as reference? When is an assay suitable for official approval? Sometimes the answer is straight forward, sometimes not. In the case of genetic typing assays gene sequencing gives the exact answer and can be used on a representative number of isolates from different host species and /or geographical origin. Typing based on antibodies often gives no similar exact results and will have to be based on emperical testing. In such cases, one strategy could be to send out the assay reagents including reference virus isolates to a number of fish disease laboratories for testing and evaluation on their respective collections of viruses. The same should be done for immunoassays to be used for diagnostic identification of viruses. Without such testing, chances of misleading results will be high, as illustrated by an example presented by Dr. Niels Jørgen Olesen (Aarhus, Denmark). The European Reference Laboratory had evaluated a commercial kit collection for the detection of a selected panel of fish pathogenic viruses including VHSV, IHNV, IPNV, and SVCV. The kit collection, which was based on fluorescence staining of infected cell cultures in tissue culture plates, fulfilled the promised requirements for the IHN kit only, whereas the IPN, the SVC and especially the VHS kit were insufficient as regards to either specificity or sensitivity.

6 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 163 Super-sensitive molecular diagnostic techniques such as PCR can be valuable and powerful, but can ironically also raise more questions then answers, as initially stressed by E- M. Bernoth (see also Bernoth, 1999). What should be concluded if the sensitivity of the methods is so high that sporadic positives are obtained, no clinical signs are observed, and no pathogen can be isolated, since it cannot grow in cell culture? A government officer, who is not always recruited from the scientific field, has to rely on field validation. This means that the researchers and the commercial companies need to understand their responsibility regarding proper validation before market release. Since it is difficult to use less sensitive assays as reference, intensive empirical testing in several laboratories including appropriate positive and negative controls will be needed before approval of such assays for e.g. surveillance programs. Otherwise we will end up with the possibility of judging fish to be virus-positive without the presence of clinical disease or the ability to confirm the presence of virus by other accepted methods. Such findings can have substantial effects on both fish-farms and disease control programs. At the end of the workshop, it was mentioned that the diagnostic reagents developed during the FAIR CT project would be available for research and testing as soon as they have been published in a research journal. Acknowledgments The authors would hereby like to thank all participants at the workshop, especially the contributors (ordered alphabetically): S. Bergman (Federal Research Centre for Virus Diseases of Animals, Institute for Infectiology, Insel Riems - Germany), E.M. Bernoth (Department of Agriculture, Fisheries and Forestry, Canberra - Australia), J-P Enzmann (Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany), K. Kippasto (Åbo Akademy Univerity, Åbo - Finland), M. Koutna (Veterinary Research Institute, Brno - Czech Republic), N.J. Olesen (Danish Veterinary Laboratory, Aarhus - Denmark) and M. Snow (FRS Marine Laboratory, Aberdeen, Scotland - United Kingdom). The workshop is a result of the EC FAIR CT project on Diagnostic methods and reference panel of reagents for detection and typing of fish viruses. References Arakawa,C.K., Deering,R.E., Higman,K.H., Oshima,K.H., O Hara,P.J., & Winton,J.R. (1990) Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus. Dis.aquat.Org., 8, Basurco,B., Vende,P., Monnier,A.F., Winton,J.R., de Kinkelin,P., & Benmansour,A. (1995) Genetic diversity and phylogenetic classification of viral hemorrhagic septicemia virus (VHSV). Vet.Res., 26, Batts,W.N., Arakawa,C.K., Bernard,J., & Winton,J.R. (1993) Isolates of viral hemorrhagic septicemia virus from North America and Europe can be detected and distinguished by DNA probes. Dis.aquat.Org., 17, Benmansour,A., Basurco,B., Monnier,A.F., Vende,P., Winton,J.R., & de Kinkelin,P. (1997) Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral haemorrhagic septicaemia

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