Indice	Molecular diagnosis of porcine viral diseases.
Indice	Sándor Belák The National Veterinary Institute, Department of Virology, Ulls väg 2B, SE-756 89 Uppsala, Sweden Address: E-mail: sandor.belak@sva.se, Tel: 0046 18 674 135 Abstract New molecular methods have been developed for the diagnosis of viral diseases of swine. Our group started the development and application of the polymerase chain reaction (PCR) for routine diagnosis as early as in 1987-88. During the last one-and-a-half-decade more than 60 PCR assays have been developed to detect a variety of DNA and RNA viruses. False positives and negatives are avoided by the use of special tools, practices and internal controls of amplification (mimics). Recently, real-time PCR methods (TaqMan, Molecular Beacons, Primer-Probe Energy Transfer System) have been developed, i.e., for the detection and differentiation of swine vesicular virus and vesicular stomatitis virus. Multiplex PCR packages are under developed for the simultaneous detection of eight important viruses of swine. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the diagnostic procedures become rapid, robust and automated. In order to standardise the real-time PCR assays, the rules of the Office International des Epizooties (OIE), are followed and the five steps of standardisation and validation are introduced. By this way the new diagnostic procedures will be nationally and internationally standardised and harmonised. Introduction Various molecular methods are under development today, in order to accelerate and improve the diagnosis of infectious diseases in animals and in man. The new assays provide possibilities for a very rapid diagnosis, since the detection of viruses can be completed within hours or even minutes. Concerning direct detection, various molecular methods are introduced, like classical-PCR, real-time PCR, multiplex PCR, improved in situ hybridisation, in situ PCR. Methods of PCR-robotics, PCR for protein detection (DNA tags), improved sample enrichment, amplification without thermocycling, macro- and microarrays are under development. Concerning indirect detection of infectious agents, ranges of other molecular methods are under construction. Portable PCR machines, pen-side tests (like dip-sticks) are developed. By following the OIE rules, international consortia are working on the five steps of standardisation, including "ring tests". In this paper we summarise the developments and experiences of our group in the field of direct detection of porcine viruses by using genetic-based systems, with special regards to the different variants of the PCR. The first generation PCR The first assays were established at our laboratory soon after the invention of the PCR, we used the method already in 1987-88 (Belák et al., 1989). Subsequently, more than 60 new PCR assays have been developed for routine diagnostic use (Belák et al., 1993a; 1993b; Belák and Thorén, 2001). First, "simple" PCR assays were used, which were soon replaced by semi-nested or nested systems, providing higher specificity and sensitivity (Belák and Thorén, 2001). Several nested PCR systems for the diagnosis of swine diseases (the amplified region in brackets): DNA-viruses: Porcine parvovirus, PPV (VP2), Porcine adenoviruses (hexon), Aujeszky’s disease virus, ADV (gB, gE, gD), Porcine cytomegalovirus (DNA polymerase). RNA-viruses: Classical swine fever virus, CSFV (NS3, E2), General pestivirus assay (5’NCR), Porcine respiratory and reproductive syndrome virus, PRRSV (ORF1b), Swine vesicular disease virus, SVD (3D), Transmissible gastroenteritis virus, TGEV (ORF7b), Encephalomyocarditis virus (PP). The sensitivity of the nested PCR systems is very high, 1-10 genome copies of the target viruses are detected. Simultaneously, the nested PCR assays provide very high specificity. In general, the systems amplify entirely the target virus(es). However, wide-range assays can also be produced. Such are the General pestivirus assays, which detect all three pestiviruses in swine, e.g., CSFV, bovine viral diarrhoea virus and border disease virus. The general assay is useful to screen for the presence of pestiviruses and then specific PCR assays determine the identity of the given pestivirus in the swine populations. PCR assays for phylogeny When examining the genetic relatedness of various viruses, the purpose is not to achieve a wide-range detection, but to obtain a high phylogenetic resolution. To provide high phylogenetic resolution, the variable genomic regions of the viral genomes are targeted. Such phylogeny PCR assays were developed for example to group pestiviruses (3’NCR, 5’NCR, E2, NS2, etc) (Vilcek and Belák, 1996; Vilcek et al., 1996; Vilcek and Belák, 1998; Björklund et al., 1998; 1999; Paton et al., 2000b). Molecular epizootiology The PCR assays of high phylogenetic resolution are useful tools not only to determine the phylogenetic relations, but also for the rapid identification the various virus variants. The genetic identification is very exact and rapid (several days or hours) and this information can be crucial during various outbreaks. The spread of the virus variants can be traced and the ways of spread may be cut rapidly, in order to prevent the distribution of the virus to large geographic areas. The rapid phylogenetic identification and tracing of the viruses is termed "molecular epizootiology". Such studies were conducted, when genetic variant of CSFV were identified in several countries of Central Europe (Stadejek et al., 1998). Viral phylogeny and molecular epizootiology were also applied in a recent study when variants of PRRSV were sequenced and characterised. We determined ORF5 sequences, representing pathogenic field strains from Poland and Lithuania, and currently available European-type live PRRSV vaccines. In addition, the complete ORF7 of Lithuanian and Polish strains was sequenced. The results showed that Polish, and in particular Lithuanian, PRRSV sequences were exceptionally different from the European prototype, the Lelystad virus, and in addition showed a very high national diversity. While all sequences were clearly of European type, inclusion of the new Lithuanian sequences in the genealogy resulted in a common ancestor for the European type virus significantly closer to the American-type PRRSV than previously seen. These observations provide support for the hypothesis that the EU and US genotypes of PRRSV evolved from a common ancestor (Stadejek et al., 2002). Development of real-time PCR assays The real-time PCR assays provide novel rapid means of virus detection in the diagnostic laboratories (McGoldrick et al., 1998; Rasmussen et al., 2003). Only one primer pair is used, but the system may still provide as high sensitivity level as the classical nested PCR. The risk of contamination is lower, since the system is closed and no sample-manipulation is needed. The contamination risk is further decreased by the fact that the fluorescence, indicating the results, is directly read through the unopened lid of the reaction vessel. Thus, there is no need to open the reaction vessels and there is no post-PCR manipulation to visualise the products. These procedures result in greatly reduced hands-on time, compared to previous PCR methods, where the products were run on agarose gels and the stained for visualisation. The single-run amplification the 96-well microtitre plate format allows automation. The diagnostic work can further be automated by using robotics for nucleic acid extraction and pipetting. Compared to previous amplification assays, the real-time PCR has a further advantage: it allows running quantitative PCR. Thus, the diagnostic answer is not only "yes" or "no", but even the amount of the of the viral nucleic acids is determined, allowing calculations to estimate the viral load during infection. Such estimation opens new path not only for the diagnosis, but also for studying pathogenesis. The estimation of quantitative aspects is crucial when a virus commonly found in animals is possibly causing symptoms in relation to viral load, for example feline coronaviruses or porcine circovirus 2 (PCV2; Krakowka et al., 2000; Liu et al., 2000). Comparative studies showed that because subclinical infections of pigs with PCV2 are common, the use of non-quantitative PCR as a diagnostic tool for PCV2-related diseases should be discouraged (McNeilly et al., 2002). The measurement of viral load is also important when estimating the effects of antiviral treatments, especially in human virology. The real-time PCR assays in routine diagnosis at our laboratory Several variants of real-time PCR methods and chemistries can be chosen today, like TaqMan, molecular beacons (MB), scorpion primers, dual probe systems as utilized in the LighCycler® (Roche), dye-labelled oligonucleotide ligation (DOL), Primer-Probe Energy Transfer System (PriProET), etc. Our laboratory has developed diagnostic TaqMan and MB and adapted PriProET systems. These assays were selected because a) reliable information was available for technical details; b) these systems seemed to be less sensitive for mutation rates of RNA viruses (an aspect important in diagnosis); c) the easy adaptability to our real-time PCR equipments, an ABI PRISM 7700 Sequence Detector (Applied Biosystems) and Rotor-Gene 2000 Real-Time Cycler (Corbett Research); d) relatively clear conditions of royalty and usage for diagnosis. TaqMan systems were developed at our laboratory or in collaboration with our partners for the detection of feline coronaviruses, parvoviruses, feline leukaemia virus, lactate dehydrogenase elevating virus, lymphocytic choriomeningitis virus, equine influenza, bovine coronavirus, bovine respiratory syncytial virus and equine rhinovirus. Furthermore, a "general" pestivirus TaqMan assay is also used (Belák and Thorén, 2001). MB assays were developed for swine vesicular disease virus and vesicular stomatitis virus, Indiana and New-Jersey serotypes (Hakhverdyan et al., 2002). The detection level of these assays is around 10 genome-copies, which indicates very high analytical sensitivity. The assays were adapted for use in routine detection of viruses and they allow diagnosis, within approximately four hours. Several examples considering emerging and re-emerging diseases of swine: Real-time PCR assays for the detection of swine vesicular disease virus (SVDV) and vesicular stomatitis virus (VSV) using molecular beacons (MBs) A conservative region of SVDV 3D-gene and VSV L-gene has been chosen to design MB probes. Each MB was labelled with a differently coloured fluorophor: FAM for SVDV, JOE for VSV-NJ and ROX for VSV-Ind. DABCYL or Black Hole Quencher (Biosearch Technologies, Inc., USA) was used as a non-fluorescent quencher. Optimisation of the probes includes followings: a) Characterisation of the probe (calculation of the signal to background ratio and thermal denaturation profile); b) Sample dilution series; 3-5. Probe, primers and manganese titration series. An ABI PRISM 7700 Sequence Detector was used for the experiments. At present SVDV MB is the most optimised among three designed probes. During the dilution series and generation of standard curve experiments (Figs 1, 2) the probe detected even 10-6 diluted RNA templates. To develop an amplification control, cloning of SVDV PCR product is being carried out. SVDV MB probe specificity test shows successful amplification of all tested SVDV isolates, but not of human Coxsackie B5 virus (Fig. 3). VSV MBs need more optimisation experiments, especially, for VSV/NJ (Hakhverdyan et al., 2002). Fig.1 Real-time PCR detection of swine vesicular disease virus (SVDV) by using molecular beacon technique. Virus dilution series from 1:1 to 1:10-6 to generate standard curves. Fig.1 Real-time PCR detection of swine vesicular disease virus (SVDV) by using molecular beacon technique. Virus dilution series from 1:1 to 1:10-6 to generate standard curves. Fig.1 Real-time PCR detection of swine vesicular disease virus (SVDV) by using molecular beacon technique. Virus dilution series from 1:1 to 1:10-6 to generate standard curves. Multiplex PCR The principle of the multiplex PCR is to use multiple primers to allow amplification of multiple templates within a single reaction. This is a useful and practical idea for diagnostic purposes, providing the chance to detect several infectious agents in a single assay. The "classical" PCR and the real-time PCR are equally suitable for designing broad-range "multiplex" PCR assays. For example, a single nasal specimen can be tested from an animal suffering from a respiratory disease, or a single rectal swab in the case of an enteritis/diarrhoea syndrome. The real-time PCR is more suitable for multiplexing, than the classical PCR, since here the individual probes can be labelled with various fluorophores, each functioning as reporter dye for one primer-set. Since the fluorescent probes emit different colour wavelengths, the real-time PCR is en excellent tool for the development of multiplex PCR assays. Multiplexing of the "classical" PCR techniques is rather complicated, considering the large number of oligonucleotides, which might interact with each other in the same reaction mix. In contrast, the concept of real-time PCR (using only single primer pairs) provides excellent possibilities for the construction of multiplex PCR assays with many targets. EC project for the multiplex detection of eight viruses of swine (QLK2-CT-2000-00486) In this ongoing project the EC is supporting the development of multiplex PCR systems for the simultaneous detection of eight viruses of swine, including OIE List A viruses. The work is performed in collaboration of six European laboratories. The viruses to be studied are African swine fever virus (ASFV), CSFV, PRRSV, ADV, PPV, SVDV, FMDV and VSV. Multiplex PCR assays are under construction to detect clusters of viruses based on possible clinical presentation. The clusters will be: a) Respiratory (CSFV, ASFV, PRRSV, ADV); b) Reproductive (CSFV, ASFV, PRRSV, ADV, PPV); c) List A, haemorrhagic (CSFV, ASFV); d) List A, vesicular (SVDV, FMDV, VSV). Further details see on: http://www.multiplex-eu.org/ Multiplex real-time PCR to detect FMDV In a recent article the consortium of the above EC project described the development of a novel quantitative real-time PCR assay for the simultaneous detection of all serotypes of FMDV (Rasmussen et al., 2003). Robotics in nucleic acid extraction The speed and efficiency of the diagnosis is further increased by the use of a nucleic acid extraction robot. Our robot (GenoVision, Norway, today Qiagen) utilizes magnetic separation of the target molecules. We have compared the results of nucleic acid preparations of the robot with manual procedures and found the robot to be more efficient and precise. In the robot, viral nucleic acids are purified simultaneously from 48 samples and the procedure is finished within 2.5 hours. The products are clean enough to be amplified directly in the PCR. Automated diagnosis The simultaneous use of the robot and real time PCR detection provided an automated diagnostic procedure. With the introduction of a pipetting robot the automation is being now further completed. Precautions to avoid false positive results When running classical PCR, false positive results, i.e., negative samples showing a positive reaction, may occur. As a general practice today in PCR laboratories, samples and mixes are handled in laminar airflow hoods, which are decontaminated using UV-C light and bleach, help to avoid false positives. We are running a recommended laboratory practice (mix and primer preparation, sample preparation, first and second PCR) in separate laboratory locations. In addition, special tube-holders and openers were constructed to minimise the false positive PCR results (Belák and Ballagi-Pordány, 1993a, b). Internal controls to avoid false negative results Inhibitory effects of ingredients, like heparin, semen components and other sample contaminants and/or pipetting errors might lead to false negative results of the PCR. In such cases the infected samples tested as negative. To avoid such misleading results, the use of internal controls (termed "mimics") is recommended. The mimics are safe indicators of amplification efficiency (Belák and Thorén, 2001). Controls in real-time PCR When running real-time PCR assays, it is also important to incorporate internal controls. A practical approach is when a selected fragment of the host animal genome is co-amplified as an internal control. By including such an intrinsic control with its specific reporter fluorophor we obtain information on the sample quality and on pipetting errors. Simultaneously, the system shows the amplification of the target nucleotide sequences and provides safety for the diagnosis. Validation, standardisation Both national and international authorities require rigorous proof today that the diagnostic assays are as reliable as possible. International agencies like OIE, FAO/IAEA, national research institutions and commercial companies make great efforts to agree on international standardisation (Wright and Zhou, 1999; Willis, 2000). Considering these requirements, our laboratory (with our partner institutions in Europe) has started the validation and standardisation of the routine diagnostic PCR assays. Diagnostic assay validation To make predictions about the performance of a diagnostic method, it is necessary to validate the assay in question. Validation is the evaluation of the method with the purpose to determine how fit the assay is for a particular field of use. General requirements for the competence of testing and calibration laboratories (EN ISO/IEC 17025:2000) This standard gives directives for an accredited laboratory and it specifies many important parameters in such an environment. It is stated that the laboratory should validate: non-standardized methods, in-house developed methods and standardized methods if they are used outside the original area of use. Examples of estimated parameters can be: LoD (Level of Detection), linearity of the method, reproducibility, repeatability, robustness or any other parameter interesting to the customer. The OIE principles of validation OIE has published (2000) a standard for the validation of diagnostic assays in the veterinary field. Chapter I.3 in this standard ("Principles of validation of diagnostic assays for infectious disease") describes in detail how to perform validation of the diagnostic assays in a standardised way. The five main stages of validation: Stage 1. Feasibility studies Stage 2. Assay development and standardisation Stage 3. Determining assay performance characteristics Stage 4. Monitoring validity of assay performance Stage 5. Maintenance and enhancement of validation criteria For details see Belák and Thorén (2001). When the assay is accepted as a routine test it is important to maintain the quality control. Consistent monitoring for repeatability and accuracy is necessary. Reproducibility between laboratories (ring tests) is recommended by OIE to be estimated at least twice a year. With our partner laboratories we have performed series of ring test to validate the PCR assays for the detection of CSFV (Paton et al., 2000a, c). If the assay is to be applied in another geographic region, it might be necessary to revalidate it under the new conditions. It is important to note that the proper assay validation is time consuming and expensive. It is difficult to obtain suitable standards and huge amounts of samples are required. This is one of the reasons that validation has been neglected or at least considered less important in the past. We can now see a clear trend that the same quality demands that have been used in human applications are now being introduced in veterinary diagnostics. Acknowledgements These studies have been carried out with financial support from the European Commission (QLK2-CT-2000-00486). References Belák, S., Ballagi-Pordány, A., Flensburg, J. Virtanen, A. (1989). Detection of pseudorabies virus DNA sequences by the polymerase chain reaction. Arch. Virol. 108, 279-286. Belák, S., Ballagi-Pordány, A. (1993a). Application of the polymerase chain reaction in veterinary diagnostic virology. Vet. Res. Comm. 17, 55 —72. Belák, S., Ballagi-Pordány, A. (1993b). Experiences on the applicability of the polymerase chain reaction in a diagnostic laboratory. Mol.Cell. Probes 7, 241-248. Belák, S., Thorén, P. (2001). Molecular diagnosis of animal diseases. Expert Review of Mol. Diagnostics 1, 434-444. Björklund, H.V, Stadejek, T., Vilcek, S., Belák, S. (1998). Molecular characterization of the 3’ noncoding region of classical swine fever vaccine strains. Virus Genes 16, 307-312. Björklund, H., Lowings, P., Stadejek, T., Vilcek, S., Greiser-Wilke, I., Paton, D., Belák, S. (1999). Phylogenetic comparison and molecular epidemiology of classical swine fever virus. Virus Genes 19, 189-195. Hakhverdyan M, Thorén P, Belák S. (2002). Real-time PCR assays for the detection of swine vesicular disease virus and vesicular stomatitis virus using molecular beacons. XII International Congress of Virology, IUMS, The World of Microbes, 27th July-1st August, 2002, Paris, p.228. Krakowka S, Ellis JA, Meehan B, Kennedy S, McNeilly F, Allan G. (2000). Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Vet. Pathol. 37, 254-263. Liu Q, Wang L, Willson P, Babiuk LA. (2000). Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. J. Clin. Microbiol. 38, 3474-3477. McGoldrick, A., Lowings, P., Ibata, G., Sands, J., Belák, S., Paton, D. (1998). A novel approach to the detection of classical swine fever virus by RT-PCR with flurogenic probes (TaqMan). J. Virol. Methods 72, 125-135. McNeilly F, McNair I, O'Connor M, Brockbank S, Gilpin D, Lasagna C, Boriosi G, Meehan B, Ellis J, Krakowka S, Allan GM. (2002). Evaluation of a porcine circovirus type 2-specific antigen-capture enzyme-linked immunosorbent assay for the diagnosis of postweaning multisystemic wasting syndrome in pigs: comparison with virus isolation, immunohistochemistry, and the polymerase chain reaction. J. Vet. Diagn Invest, Mar. 14(2), 106-112. Paton, D.J., McGoldrick, A., Belák, S., Mittelholzer, C., Koenen, F., Vanderhallen, H., Biagetti, M., De Mia, G-M., Stadejek, T., Hofmann, M., Thuer, B. (2000a). Classical swine fever virus: a ring test to evaluate RT-PCR detection methods. Vet. Microbiol. 73, 159-174. Paton, D., McGoldrick, A., Greiser-Wilke, I., Parchariyanon, S., Song, J.Y., Liou, P.P., Stadejek, T., Lowings, P., Björklund, H., Belák., S. (2000b). Genetic typing of classical swine fever virus. Vet. Microbiol. 73, 137-157. Paton, D.J., McGoldrick, A., Bensaude, E. Belák, S., Mittelholzer, C., Koenen, F., Vanderhallen, H., Greiser-Wilke, Ischreibner, H., Stadejek, T., Hofmann, M., Thuer, B. (2000c). Classical swine fever virus: the second ring test to evaluate RT-PCR detection methods. Vet. Microbiol. 77, 71-81. Rasmussen, T., Uttenthal, Å., de Stricker, K., Belák, S., Storgaard, T. (2003): Development of a novel quantitative real-time PCR assay for the simultaneous detection of all serotypes of foot-and-mouth disease virus. Archives of Virology, 148. 2005-2021 Stadejek, T., Vilcek, S., Lowings, P., Ballagi-Pordány, A., Paton, D., Belák, S. (1998). Genetic heterogeneity of classical swine fever virus in Europe. Virus Res. 52, 195-204. Stadejek, T., Stankevicius, A., Storgaard, T., Oleksiewicz, M. B., Belák, S., Drew, T. W., Pejsak, Z. (2002). Identification of radically different variants of porcine reproductive and respiratory syndrome virus (PRRSV) in Eastern Europe: Towards a common ancestor for European and American viruses. J. Gen. Virol. 83, 1861-1873. Vilcek, S., Belák, S. (1996). Genetic identification of pestivirus strain Frijters, isolated from pigs, as a border disease virus. J. Virol. Methods 60, 103-108. Vilcek, S., Stadejek, T., Ballagi-Pordány, A., Lowings, J.P., Paton, D.J., Belák, S. (1996). Genetic variability of classical swine fever virus. Virus Res 43, 137-147. Vilcek, S., Belák, S. (1998). Classical swine fever virus: Discrimination between Vaccine strains and European field isolates by restriction endonuclease cleavage of PCR amplicons. Acta Vet. Scand. 39, 395-400. Willis NG. The role of the OIE in international trade. (2000). Ann. N .Y. Acad. Sci. USA. 916, 1-5. Wright P, Zhou EM. Developments in international standardization. (1999). Vet. Immunol. Immunopathol. 72, 243-8.
Arriba	© www.exopol.com - mail