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Belak thorenleblancviljoenreview2009

  1. 1. Review Advances in viral disease diagnostic and molecular epidemiological technologies Expert Rev. Mol. Diagn. 9(4), xxx–xxx (2009)Sándor Belák†, Peter The early and rapid detection and characterization of specific nucleic acids of medico–veterinaryThorén, Neil LeBlanc pathogens have proven invaluable for diagnostic purposes. The integration of amplification andand Gerrit Viljoen signal detection systems, including online real-time devices, have increased speed and sensitivity† Author for correspondence and greatly facilitated the quantification of target nucleic acids. They have also allowed forJoint Research and Development sequence characterization using melting or hybridization curves. The newer generation ofDivision, Department of molecular diagnostic technologies offers, hitherto, unparalleled detection and discriminationVirology, Swedish University of methodologies, which are vital for the positive detection and identification of pathogenic agents,Agricultural Sciences and as well as the effects of the pathogens on the production of antibodies. The development phaseNational Veterinary Institute, of the novel technologies entails a thorough understanding of accurate diagnosis andThe Collaborating Center for the discrimination of present and emerging diseases. The development of novel technologies canBiotechnology-based Diagnosis only be successful if they are transferred, and used, in the field with a sustainable quality-assuredof Infectious Diseases in application to allow for the optimal detection and effective control of diseases. The aim of theseVeterinary Medicine of the OIE new tools is to detect the presence of a pathogen agent before the onset of disease. This(World Organisation for Animal manuscript focuses mainly on the experiences of two World Organisation for Animal HealthHealth), Ulls väg 2B,SE-756 89 Uppsala, Sweden collaborating centers in context to molecular diagnosis and molecular epidemiology ofTel.: +46 1867 4135 transboundary and endemic animal diseases of viral origin, food safety and zoonoses.Fax: +44 870 136 3214 Keywords : amplification chemistry • detection chemistry • early and rapid diagnosis • microarraysandor.belak@bvf.slu.se • molecular epidemiology • PCR • phylogeny • probe • label • transboundary animal disease • viral disease • viral metagenomic Secure and safe food, an increasing human have contributed to new and challenging high- population, an increase of human and animal risk situations. The recent worldwide incidences movement, intensification of animal husbandry, of foot-and-mouth disease, the extension of blue- globalizing trade of live animals and/or animal tongue in the European region and Rift Valley products, bedding and feeds, are all factors that fever into the Arabian Peninsula, as well as the are leading to the increasing threat of emerg- recent reoccurrence and rapid spread of influ- ing and re-emerging of animal diseases (includ- enza viruses, demonstrates the serious economic ing unknown diseases) and those that affect and social impact of highly contagious trans- humans. Food security has a high public prior- boundary animal diseases (TADs). ity and the biggest threat is seen as the outbreak Global climatic changes or variations are pre- of a pandemic event. Despite our best efforts dicted to have a direct effect on the occurrence, with regards to intensive disease control and emergence and spread of viral diseases, especially progressive vaccination programmes, the imple- of those that are transmitted by insect vectors. mentation of early-warning tools and systems, This predicted trend is clearly illustrated by the the use of diagnostic programmes and tools, the northward spread of bluetongue virus [3] and utilization of marker and tracer vaccines [1,2] , African horse sickness virus in Europe – regions the unpopular employment of stamping-out of the world where these diseases have previously policies and the regulation of animal transfer not been reported. This phenomenon is a conse- or movements, animal diseases still have a very quence of insect vectors (Culicoides) expanding high impact on animal health and human wel- into new territories that were previously unsuit- fare. Globalization and the opening of borders able for their life-cycles. Other vector-borne viral between countries (e.g., the European continent) diseases, such as African swine fever, also show awww.expert-reviews.com 10.1586/ERM.09.19 © 2009 Expert Reviews Ltd ISSN 1473-7159 1
  2. 2. Review Belák, Thorén, LeBlanc & Viljoensimilar tendency of expansion to new, previously noninfected ter- the development of amplification platforms, such as PCR; there-ritories. In addition to TADs, there is a range of other animal dis- fore, the biased focus of this article is toward thermal amplifica-eases with a ‘more restricted’ or endemic geographic character, but tion platforms and their associated amplification and detectionwith an equally high economic and socioeconomic impact. These chemistries. However, these technologies have not yet been fullydisease pathogens (e.g., bovine herpesviruses, adenoviruses, bovine exploited and developed into applied tools. Furthermore, effi-viral diarrhea virus [BVDV], bovine respiratory syncitial virus and cient, affordable and reliable devices that combine these amplifi-bovine coronaviruses) are frequent in cattle populations through- cation technologies with sample processing through ana lysis andout the world and their elimination or eradication are important reporting processes are needed for early warnings against manyongoing activities [2,4,5] . Similarly, the swine populations of the important livestock diseases. This article is aimed at providing anworld suffer from a wide variety of viral diseases – classical swine overview of some of these technological advances.fever, African swine fever, Aujeszky’s disease, foot-and-mouthdisease and swine vesicular disease. For example, classical swine Molecular diagnosis of viral diseasesfever has been eradicated from domestic pig herds in the EU, but The development of diagnostic and monitoring tools are experi-wild boar populations are still infected in several EU countries, encing an unprecedented growth phase. This is partly owing toposing a permanent danger of reinfection for the whole continent; the increased demand for secure and safe food and on the needin addition, the spreading of African swine fever is complicated by countries to detect and control plant and animal pests andby the fact that there is no competent vaccine available and that diseases as part of their public obligation. The following sectionsnew variants of the virus are emerged in Africa, while in Sardinia, will concentrate on the molecular diagnostic and characterizationthere is an endemic form of the disease. Porcine respiratory and technologies that are showing promise.reproductive syndrome is now a commonly occurring disease,caused by an emerging arterivirus, that was first detected in 1991 Gel-based & real-time PCR platformsin The Netherlands and has since rapidly spread throughout the A wide range of PCR [6] and real-time PCR assays [7] have beenworld. There are other diseases of growing importance, such as developed since the late 1980s and applied to the diagnosis ofpostweaning multisystemic wasting syndrome or porcine derma- TADs and other infectious diseases, as well as for the improvedtitis and nephropathy syndrome, that are associated with porcine detection of pathogens in human food and animal feed. Real-timecirvovirus type 2; however, several questions are still unanswered PCR has truly changed the molecular diagnostic scene and mustin etiology, pathogenesis and many other important factors of be considered as one of the more important developments dur-these swine diseases. In addition to the aforementioned examples ing the last few years. The various real-time PCR platforms andgiven for livestock, there is a long list of emerging or re-emerging their respective chemistries, such as Taqman®, molecular beacons,diseases in various host species, including humans, such as the primer probe energy transfer (PriProET), scorpion primers, dualdiseases caused by hantaviruses, Japanese encephalitis virus, HIV, probe systems, such as those utilized in the LightCycler® (Roche,dengue viruses, menangle virus, Australian bat lyssavirus, ebola NJ, USA), dye-labeled oligonucleotide ligation and SYBR Green,virus, avian influenza variants, such as H5 and H7, severe acute have showed high sensitivity and specificity in the detection ofrespiratory syndrome (SARS), coronavirus, nipah virus and hen- viral and bacterial pathogens [6–12] . Compared with the ‘classical’dra virus, amongst others, which have recently caused concern. single, nested or ELISA PCR platforms, real-time PCR assaysSeveral of these diseases have serious zoonotic concerns and global offer a number of important, well-known advantages:implications. This list clearly indicates that there is a need for the • Faster and higher throughput assays;following: • One-step amplification set-up that provides sensitivity and• Early and confirmatory detection of harmful pathogens specificity close to or equal to traditional nested PCR;• Improved diagnostic technologies for both human and animal • Detection of amplified products measured online and in real- populations time through the lid, side or bottom of the reaction vessel with-• Harmonization and validation of such technologies out opening the system, thus minimizing the risk of contami- nating the laboratory environment, or carryover into nearby At this point, it is important to note that there exist many com- samples;petent diagnostic assays that are fit for their intended purpose butmight, in varying degree, need to be validated and harmonized. • Post-PCR handling of the amplicons/products is not required To facilitate the timely diagnosis and effective control of TADs (i.e., the contamination risk posed by postamplification prod-and those of zoonotic nature, scientists and private enterprises ucts are minimized);have embarked on a research-and-development path in search • Output is not only ‘positive’ or ‘negative’, but allows for a quan-of molecular tools that will be able to satisfy the need for the titative estimation of target or input nucleic acid in the sample;early and rapid detection of harmful pathogens. Critical tech-nologic advances for the rapid detection and diagnosis of high- • Hands-on time is greatly reduced, compared with traditionalthreat diseases are becoming increasingly available in research detection using agarose gels followed by ethidium bromide orand industrial laboratories. Much emphasis has been placed on GelRedTM staining;2 Expert Rev. Mol. Diagn. 9(4), (2009)
  3. 3. Trends in viral diagnosis & epidemiology Review• Practical to automate with the use of a 96-well microtiter plate need to adapt assays to their own work processes. Not all labora- format, without the need for nested PCR; tories are going to use the exact same reaction conditions but it is very important that the resulting varieties of assays will result• High throughput potential due to robotic automation on both in comparable assay characteristics in each diagnostic laboratory. the sample extraction and the sample processing/amplification sides; Sensitivity & specificity of real-time PCR platforms• Probes can be labeled with a number of different fluorophores Some real-time PCR platforms are able to detect as few as ten that function as individual reporter dyes for different primer genome copies of the targeted viruses. This indicates very high sets. Thus, it is suitable for the development of multiplex PCR analytical sensitivity and specificity, with the majority of the plat- systems; forms being able to exclusively detect and amplify the selected target nucleic acids where crossreactivity could be a factor in the• Lower costs per detected agent, if the equipment can be used diagnosis. However, ‘wide-spectrum’, ‘pan-‘ or ‘general’ PCR for a large number of samples. For further information see [7,8] . assays, for example the ‘pan-pesti’ PCR assays, which amplify The advantage of real-time PCR techniques in allowing the very conservative regions of the pestivirus genomes (e.g., selectedquantitative assessment of the targeted genomes can be very regions from the 5´NCR) and are able to amplify all tested pes-important in disease diagnosis, such as in the case of diagnosis of tiviruses, such as BVDV, classical swine fever virus (CSFV) andpostweaning multisystemic wasting syndrome where the number border disease virus [11] are useful where a wider range of targetsof viral particles or the viral loads of porcine circovirus type 2 have can be expected or the exact causative agent is not known [18] .to be determined [13] . Currently, TaqMan and molecular beacons One strategy of laboratory diagnostic ana lysis could be to useare the most commonly used real-time PCR chemistries in routine an arsenal of wide-range (general) real-time PCR assays for pre-diagnostic laboratories [10] and, therefore, this article will focus liminary screening of the samples [19] as a first line of detection,mostly on these, but will include other exciting approaches, such while the exact identification/characterization of the detectedas the PriProET and light upon extension (LUX) systems [14–16] . pathogens is made by narrower-range, highly specific PCR assays. New real-time PCR applications, such as the linear-after-the- These assays not only allow for the exact identification of theexponential (LATE)-PCR [6] are now considered as robust, reli- virus variant(s) but also allow for its molecular epidemiologicalable assays for improved detection of various pathogens, especially characterization [9,10] .when adapted to simple tools, such as portable PCR machines,that will allow onsite disease diagnosis [17] . These platforms will Multiplex PCR in routine diagnosisincreasingly be commercialized as early, rapid, on-site and high- Multiplex PCR platforms are based on the use of multiple primersthroughput systems. The specification of the real-time PCR to allow amplification of multiple templates within a single reac-platform, such as TaqMan or molecular beacons, is important, tion. For example, this approach can be used to analyze a singleconsidering that the various platforms and chemistries have differ- nasal swab for a respiratory disease or rectal swab collected froment strengths and weaknesses, such as detection range, sensitivity, an animal suffering enteritis/diarrhea syndrome. By performingspecificity and sensitivity to mismatch, which influence the plat- multiplex PCR, the detection specificity is broadened to diagnoseforms results. It is important to note that, even within the same all possible pathogens present that can be considered as agentsplatform of real-time PCR or similar real-time PCR chemistries, for a particular disease. These platforms are useful for diagnosticdifferences can, and will, occur within and between laboratories purposes, providing the laboratory has the capability to analyze[7] . This variation has been highlighted with various TaqMan and interpret the more complex multiplex results.assays developed for the detection of the same virus that can The gel-based PCR platforms facilitated the development ofdiffer with regards to diagnostic specificity, sensitivity and other multiplex PCR assays, but the advent of real-time PCR platformsimportant assay parameters [17,18] . Therefore, it is important to provided an important high-throughput tool in a diagnostic labo-clearly specify the exact real-time PCR platform, chemistry and ratory’s arsenal. This development was possible as all the indi-assay concerned and to provide very exact specifications for the vidual and specific probes used for the component assays canapplied method in order to avoid confusion and misunderstand- be labeled with a different fluorophores, each of which functioning between results. as a specific color reporter dye for one set of primers. Since the On the other hand, a molecular diagnostic laboratory gener- fluorescent probes emit at different color wavelengths, it enablesally has a number of assays to analyze and it is not practical if all multiplexing of the assays [10,20–23] . Compared with single-tar-assays used are of different design or utilize different real-time get PCR platforms, construction of multiplex platforms can bePCR machines. Thus, it is not feasible to believe that molecular rather complex, considering the large number of oligonucleotidesdiagnostic laboratories will set up the recommended assays in required to hybridize to different targets with different empiricexactly the same way. Some adaptation to various nucleic acid values [6] . The multiplex real-time PCR platform has the potentialextractions, real-time PCR machines or PCR kits will always be to produce considerable savings in time and effort, without com-the case, further underlying the importance of community refer- promising the robustness and sensitivity of virus detection assaysence laboratories and annual proficiency testing. It is also impor- [10] . However, the competition of ‘in-test-tube’ oligonucleotidestant to note that reference samples are available to laboratories in frequently hinders the development of potent multiplex PCRwww.expert-reviews.com 3
  4. 4. Review Belák, Thorén, LeBlanc & Viljoen microarray format using spotted glass slides has not been well A B C accepted in diagnostics laboratories and will be replaced with more user-friendly planar formats. The company, CLONDIAG Chip Technologies GmbH (Jena, Germany) has produced the ArrayTube® and ArrayStrips™ sys- tems, which use a planar array printed on the bottom of custom sample tubes or strips and read in their specialized workstations. The ArrayTube is a single tube with an array printed on the bot- tom of the tube. The ArrayStrips use the same principle but are Figure 1. Clondiag Chip technolgies ArrayTube platform. designed for use in 96-well format, suitable for automation and (A) ArrayTube (with probe array chip at the bottom of the tube), highthroughput (Figure 1) [102] . This system is in the mid-plex range, (B) the ATR 03 reader (connected to a PC or laptop to run image with a capacity of 400 probes per tube. The UK’s Veterinary acquisition and analysis) and (C) magnification of probe array Laboratories Agency (Surrey, UK) has launched a commercial chip. The detection reaction consists of horseradish peroxidase product, known as Identibac ®, for the ana lysis of antimicro- (HRP) and a HRP substrate, tetramethylbenzidine, which react to form a blue precipitate. bial resistance or virulence in a range of different bacteria [103] . In The Netherlands, the Institute for Food Safety ([RIKILT],platforms and, therefore, necessitates an optimal primer/probe Wageningen, The Netherlands) is also developing kits for routinedesign with as few crossreaction possibilities as possible. ‘Primer pathogen detection in combination with the Padlock Probe chem-competition’ could lead to a decrease in the levels of specificity and istry. Examples of this technology applied to viral diagnosticssensitivity of the PCR platform and, in certain cases, the system include the detection and subtyping of avian influenza [25] and thewill simply not work at all in a multiplex arrangement. detection of herpesvirus and adenovirus coinfections [26] . Aside A novel multiplex detection system for bacterial genomes using from planar arrays, other multiplex technologies are also available.zinc-finger proteins was developed recently [24] . Zinc-finger pro- Seegene Inc. (MD, USA; Seoul, Korea) has 16 CE marked prod-teins are DNA-binding proteins that can bind to dsDNA with ucts, most of which include viral detection. One such assay is thehigh affinity and specificity. Since zinc-finger proteins can Seeplex® RV12 Affinity Capture Elution detection assay designeddirectly detect PCR products and can double check the specific to detect 12 major respiratory viruses, 11 respiratory RNA virusesPCR amplification and sequence specificity of the PCR prod- and one DNA virus from patients’ samples, including nasopha-ucts, this novel method has great potential and has allowed the ryngeal aspirates, nasopharyngeal swabs and bronchoalveolardetection for three pathogens, Legionella pneumophila, Salmonella lavage. This technology has also been applied to the veterinaryspp. and influenza A virus, in a multiplex format. Many such field with the Seeplex® Porcine Diarr-V Detection Kit and Seeplexadvances have been published recently, and it takes a matrix of Porcine Diarr-B Detection Kit, which are designed to detect fivescientific and commercial entities to work together to ensure the viruses and bacteria playing roles in swine gastrointestinal dis-sustainable development of these platforms into the diagnostic eases, including porcine epidemic diarrhea. The general systemmarket. Advances in both hardware and molecular chemistries used for these assays combines a novel chemistry using bipartitehave allowed single-well multiplex assays to become a reality in primers connected with a poly-inosine linker and conventionalroutine laboratories, with assays that are both US FDA approved gel-based detection using amplicon size [27–31] .and Conformité Européene (CE) marked. Another multiplex system, which has entered the field of viral diagnostics, uses an analogous approach to the conventionalMicroarrays & other novel technologies microarray for detection, where nucleic acid probes are cova-The advancement in microarray technology created a break- lently linked to microspheres instead of attached to a solid sur-through in diagnostics allowing many specific sequences to be face. This technology was brought to the market by the Luminexanalyzed simultaneously. Microarrays function in a number of Corporation (TX, USA) and has since been licensed to manydifferent ways but the common characteristic is that many spe- other commercial partners (Figure 2) . In this system, nucleic acidscific pieces of nucleic acid can be identified through the use of can be coupled to coloured microspheres creating up to a 100-complementary probes that make up the array. In diagnostics, plex assay on the Luminex® 200™. Luminex has announced athe first microarray-based kit approved by the FDA [101] and for 500-plex instrument for release in 2009. This technology runs onuse in the EU was in 2004. The test is called the AmpliChip® an open architecture platform so assays can be custom designedCytochrome P450 Genotyping Test (Roche Molecular Systems by researchers using ‘naked beads’ and oligonucleotides of theirInc., CA, USA) and was cleared for use with the GeneChip® own design [32] . As an aside, it is noteworthy that this technologyMicroarray Instrumentation System (Affymetrix Inc., CA, USA). can also be used to detect proteins [33] . In viral diagnostics, theThe assay uses a solid-phase planar microarray format and was xTAG Respiratory Viral Panel [34] received 510(k) clearance fromdesigned to identify dozens of variants of the genes CYP2D6 and the FDA in 2008 (also CE marked for use in EU). This test wasCYP2C19 in patients to help determine their proficiency in drug FDA cleared for 12 viruses and subtypes and CE marked for 19.metabolism. Later on, multiplex and microarray-based diagnostics The xTAG® Respiratory Viral Panel is the first multiplexed nucleicexpanded into the field of viral diseases; however, the traditional acid test for respiratory viruses cleared for in vitro diagnostic use4 Expert Rev. Mol. Diagn. 9(4), (2009)
  5. 5. Trends in viral diagnosis & epidemiology Reviewby FDA. It also is the first test of any kindthe FDA has cleared to detect humanmetapneumovirus, the first test clearedfor influenza A subtyping and the first Eever molecular test cleared for adenovirusdetection. The molecular chemistry usedin this assay involves several primer pairs, C Fbipartite primers that contain complemen- Btary sequences to those on the microspheresand two rounds of PCR. A Technologies different from the probe-based methods mentioned exist and are Dbeing applied in viral diagnostics; theseinclude surface plasmon resonance andmass spectrometry. One, in particular, Figure 2. Luminex laboratory at the National Veterinary Institute and thethat has been applied to viral diagnostics is Swedish University of Agricultural Sciences in Uppsala, Sweden. Luminex 200 system, including (A) waste bottle, (B) the Luminex SD sheath fluid delivery system thethe IBIS™ system, which uses mass spec- (C) Luminex 200 instrument, the (D) Luminex XY plate-handling platform andtrometry on amplified PCR products to (E) Luminex xPONENT 3.0 software and a PC. The microsphere-based detection systemderive base compositions [35–39] . This was allows for 100 different analytes to de detected in each sample (100-plex). Also pictured,used, for example, to detect an unknown is (F) the Tecan Hydroflex wash-station with buffer and waste bottles. This instrumentcontaminant (found by IBIS system to be performs automated microplate washing and vacuum filtration for use with both bead types (magnetic and polystyrene) used in the Luminex system.bluetongue virus) in a large virus culturelibrary [Bulte r, pers . comm.] . Furthermore, high demand for these equipments, industry is producing a widelow-volume, multiwell, PCR-based systems are reaching the point range of various types of the nucleic acid-purifying robots. Aswhere automated microfludic platforms and nanovolume reaction examples, the Magnatrix 8000™ and the recent Bullet (Nordiag,mixes offers a variety of systematic options in terms of designing Norway), the Qiagen Symphony™ and others, utilize magneticfit-for-purpose molecular detection systems that generate massive separation of the target molecules and nucleic acid preparationsamounts of PCR data. , and the results are more efficient and reliable than manual pro- The BioTrove OpenArray system is an example of such a tech- cedures. The robots simultaneously purify the nucleic acids fromnology, using a standard PCR cycling protocol designed to func- 96 samples within 3–4 h and the products are clean enough to betion with the system. It can produce 3072 simultaneous PCRs amplified directly in the PCR. In addition to high speed, robust-in 33-nl reaction volumes in one plate (approximate dimensions ness and low labor input, robots also can reduce the risk of crossof conventional microarray slide). The plate composition consists contamination between specimens.of 48 subarrays, each with 64 through-holes or wells (Figure 3) . To The introduction of special tools, laboratory practices and inter-perform the PCR, the OpenArray plate can be constituted in two nal controls (mimics) made it possible to reduce the danger ofways; either the wells can be spotted with primers or the wells can false-positive and -negative results in the protocols of diagnosticbe empty with primers being added as part of the reaction mix. PCR assays [7,9,10] . Closed and automated systems of extractionSample application to the wells can be configured in a variety of robots provide internal security for the PCR-based diagnosticways with the assistance of the AutoLoader equipment. The NT assays, as human manipulations are kept to a minimum within thecycler can run three plates simultaneously (9216 individual PCR assay. It is possible to automate most steps in diagnostic proceduresassays) and monitors the reaction progress in real-time. using nucleic acid extraction and pipetting robots, followed by PCR in real-time PCR machines, thus establishing an automatedRobots accelerate molecular diagnosis & improve safetyAn important aspect of the molecular diagnostic process is theextraction of nucleic acid [6] . This step has a large impact on thedown-stream applications and can be very difficult depending onthe origin of the sample. Several sample types are prone to inhibitPCR and could have profound impact on the outcome of thediagnostic procedure. In addition, RNA/DNA extraction couldbe a real bottleneck when large numbers of samples are processed.The use of nucleic-acid extraction robots is now common in the Figure 3. BioTrove OpenArray platform plate. A platepreparation and purification of the targeted viral nucleic acids consists of 48 subarrays each containing 8 × 8 through-holesthat are crucial steps of molecular diagnostic procedures. The (wells) for a total of 3072 reaction chambers per plate. Threeuse of these extraction robots has accelerated the diagnostic pro- plates can be read in the NT instrument (not shown) for a total ofcedures, improved repeatable and increased safety. Realizing the 9216 simultaneous real-time qPCR results.www.expert-reviews.com 5
  6. 6. Review Belák, Thorén, LeBlanc & Viljoen operators require no technical understanding of the PCR meth- A B odologies but the equipment provides an outcome concerning the suspected diagnosis of a viral disease in the field (Figure 4) [104] . Detection is based on advanced PCR chemistry known LATE PCR. This is an advanced form of asymmetric PCR that efficiently generates single-stranded amplicons under controlled conditions. This technology also has improved sample prepara- tion, suppressed amplification errors, improved probe design for rapid high-resolution ana lysis of the amplified product to make multiplexing easier that allows for rapid DNA sequencing and enhance data ana lysis [42,105] . This combination of a simple, por- table PCR machines and LATE-PCR technology provides simple facilities for the on-site diagnosis of viral diseases. Figure 4. BioSeeq Portable Veterinary Diagnostic System. (A) Portable PCR instrument with five independent thermocyclers (running assay in far left thermocycler) with built in Molecular epidemiology communications, including GPS, WiFi and Bluetooth technology. PCR assays yield specific DNA products or amplicons that can be (B) Disposable automated sample preparation unit processes raw analyzed, sequenced and characterized by several means. Nucleic sample into PCR mixture allowing test to be conducted at acid sequences can be analyzed and compared with each other pen-side. and with previously described sequences, obtained from large international databases, such as GenBank. The rapid nucleic aciddiagnostic chain. The introduction of robots has led to the estab- phylogenetic identification, classification and tracing of DNA andlishment of high-throughput and robust diagnostic assays with RNA targets (in this case, viruses) is termed ‘molecular epidemiol-less contamination risk and shortened diagnosis time from hours ogy’. Studies on molecular epidemiological were conducted, forto minutes [9] . Nucleic acid-based diagnosis has developed along example, when genetic variants of CSFV were identified in severalsimilar lines to that of ELISA-based diagnostic chains, using auto- countries of Central Europe. It was hypothesized that Europeanmated systems that provide rapidity, robustness, low costs, reduced and US genotypes of the porcine respiratory and reproductive syn-labor-requirement and an increased reliability of diagnosis. drome virus evolved from a common ancestor that was suspected to have originated from Eastern Europe [43–46] .Isothermal amplification using simple thermoblocks PCR amplification and comparative nucleotide sequence ana-Alternative methods, such as Invader® or loop-mediated isother- lysis not only allows for the direct detection of the viruses but alsomal amplification (LAMP) technologies, utilizing the isothermal retrospective genetic ana lysis of biological products and clinicalcharacterization of the amplifying mechanism instead of varying samples. Such approaches can also be applied to determine thetemperature amplification methods (our commonly used PCR identity of virus strains used in vaccine production. Recent studiestechnology) are becoming more accepted. In short, LAMP is very have found that a virus ‘pick up’ had occurred in a commerciallysimple and rapid wherein the amplification can be completed in produced, live-attenuated BVDV vaccine. These results empha-less than 1 h under isothermal conditions employing a set of six size that the contamination of commercially available live vaccinesspecially designed primers spanning eight distinct sequences of a with exogenous virus strains (e.g., the BVDV strain originatingtarget gene by incubating all the reagents in a single tube. These from fetal calf serum or bovine cells) is a real risk factor in bioin-alternative systems do not need the costly investment of PCR- dustries. Unequivocal ana lysis, including molecular methods, isamplifying machines as isothermal amplification methods use needed to verify the authenticity of biological products, such assimple thermoblocks that are more affordable [40,41] . vaccines, fetal calf serum batches and cell lines, to reduce the risk of viral contamination [47] . In addition, molecular epidemiologyPortable PCR machines can be a useful tool for animal health authorities when combat-Portable PCR machines are constructed to bring laboratory facili- ing various viral diseases and when doing their risk assessment.ties closer to field cases and disease outbreaks [17] . Several com- The occurrence of virus variants can be detected; and the spreadpanies are producing and optimizing battery-powered machines of various variants traced, allowing epidemiological ana lysis andthat can be easily used under field conditions, which allow for provide the necessary support to combat infection and controlcomplete disinfection of the equipment, require simple sample disease spread.preparation and produce rapid results without the need for specifictraining. For example, Smiths Detection’s Portable Veterinary World Organisation for Animal Health standards forDiagnostic Laboratory takes the laboratory to the field. It has international standardization & validation of PCR-basedbeen designed with field veterinarians in mind, comprising of a diagnostic assaysportable briefcase-sized PCR instrument and a disposable sample Considering the frequent occurrence of viral diseases world-preparation unit. This integrated system provides rapid on-site wide and the intensive research and development in the field ofidentification under a wide range of weather conditions. The molecular diagnosis, there is an ongoing need for international6 Expert Rev. Mol. Diagn. 9(4), (2009)
  7. 7. Trends in viral diagnosis & epidemiology Reviewstandardization and validation of developed assays. It is critical target/probes are then amplified with a single universal primerthat veterinary diagnostic laboratories use validated techniques set. The high throughput sorting of fluorescently labeled ampli-fit for their intended purpose to provide comparable results that cons are then sorted, using the tag sequences, on a microarrayallow the same conclusions and actions to be taken in different platform. Although the cost for oligonucleotides and array slidesregions of the world. This consolidated and integrated approach is higher than that for conventional PCR assays, it is reducedis seen as having the best chance of success to combat TADs in proportion to the number of assays in comparison with con-and endemic diseases effectively on a wider or global scale while ventional PCR assay. In this format, multiplex detection usingadhering to the ‘one world, one health’ principle. padlock probes and microarrays could have implications in more- National and international authorities require rigorous proof effective screening for viruses causing similar vesicular symptomsthat assays used in laboratories are as reliable as possible and and, in turn, facilitate rapid counteractions, especially in case ofprovide identical results [48] . International agencies, such as the foot-and-mouth outbreaks [49] .World Organisation for Animal Health (OIE), the Joint Food andAgriculture Organization/International Atomic Energy Agency Padlock probes for avian influenza viruses(FAO/IAEA) Division, national research institutions and com- An assay has been developed for the simultaneous detection andmercial companies take considerable effort to provide guidelines subtyping of avian influenza virus (AIV) using padlock probeon international standardization of protocols. The OIE regu- chemistry for multiplexed preamplification and microarray detec-larly publishes standards for the validation of diagnostic assays tion. The assay has the ability to identify both the hemagglutinin[106] . Validation and international standardization of nucleic acid and neuraminidase surface antigens of AIV in a single reaction.amplification-based diagnostic methods (e.g., PCR) is a major Gyarmati et al. analyzed 77 influenza strains, representing thetask for the animal-health authorities. The in-house PCR assays entire assortment of hemagglutinin and neuraminidase, and allwill soon have to be replaced by validated and standardized inter- (77 out of 77) of the samples were identified as AIV and 97%national procedures or be properly validated themselves (please see (75 out of 77) were correctly subtyped [50] . The specificity ofthe OIE Manual of Diagnostic Tests and Vaccines for Terrestrial the assay was determined testing heterologous pathogens. TheAnimals 2008 [107]) . results indicated that the assay is a useful and robust tool with a high throughput for the rapid detection and typing of AIVsRecent developments in the field of compared with conventional methods. In summary, the padlockmolecular diagnostics probes, adapted to microarray formats, provides a novel means forImproved detection of foot-and-mouth disease virus powerful and very complex novel molecular diagnosis. ComparedIt is important to mention foot-and-mouth disease, as a trade- to real-time PCR assays, the padlock probe-based microarraysrelated disease, emerges and re-emerges in areas where it is con- are inferior in sensitivity, but they allow for a highly multiplexsidered eliminated or where it did not occur before. A character- diagnosis and simultaneous ana lysis of thousands of specimensistic of this virus is its genetic and antigenic drift. It is, therefore, in the same system.important to be able to detect and differentiate the appropriateserotype in the field if possible. For this purpose, a robust quan- Novel TaqMan® & primer-probe energy-transfer assaystitative multiplex real-time PCR platform for the simultaneous for the detection of hepatitis E virusdetection of all the seven serotypes of foot-and-mouth disease Hepatitis E virus is an important cause of food- and waterbornevirus (FMDV) has been developed. This platform is based on diseases in countries with poor sanitation, but recently it has beenthe proven PriProET principle and is targeting the coding region occurring more frequently in regions of the world where healthof the 3D gene of the viruses. The authors provided sufficient services are at a high standard. Previously, the disease cases weredata to conclude that a complex diagnosis of a foot-and-mouth associated with traveling but recently zoonotic transmission hasdisease outbreak can be accomplished within a short period of also been suspected (i.e., a direct route of infection from animalstime [14] . to humans). Two real-time PCR methods have been developed and compared: a TaqMan® and PriProET assay to improve theSolid-phase microarrays in veterinary diagnostic virology diction of the virus. These robust, highly sensitive methods pro-based on padlock probes vide valuable diagnostic tools to investigate zoonotic transmissionA solid-phase microarray system has been developed for the and to detect the virus in the food chain. They have also been usedsimultaneous detection of FMDV, vesicular stomatitis and in research related to the potential of hepatitis E virus to crossswine vesicular disease viruses using padlock probes [49] . The the species barrier. The two novel PCR assays detected a broadapplication of padlock probes to the detection of pathogens is range of viruses representing all the four genotypes of hepatitis Ea recent development in molecular disease diagnosis. Padlock virus. TaqMan assay showed higher fluorescence values for posi-probes have the capacity to detect many different nucleic acid tive samples, while the PriProET assay better tolerated the pointtarget sequences in a single multiplex array platform system. mutations in the target nucleic acids and, therefore, is a moreEach viral nucleic acid template is a target for a specific and powerful tool for the detection of new hepatitis E virus variants.unique padlock probe [49,50] . The more available specific probes, These two real-time PCR assays are useful novel tools for virusthe more ‘different’ targets can be detected. The circularized detection and molecular epidemiology [51] .www.expert-reviews.com 7
  8. 8. Review Belák, Thorén, LeBlanc & Viljoen • It is an isothermal amplification method that requires a simple A B thermoblock • It is rapid, with the result being obtained within 30–60 min • It is highly specific and sensitive • The test reading is simple as the results are visualized either by gel electrophoresis or visually through the addition of SYBR Green (Figure 5) Reverse transcriptase-LAMP provides a novel tool for the Figure 5. Simple detection of swine vesicular disease virus ‘front-line’ diagnosis of swine vesicular disease, an important by loop-mediated isothermal amplification assay assay TAD since it can easily be performed in modestly equipped field with the application of isothermal amplification using only laboratories and adapted to mobile diagnostic units. a simple thermoblock. (A) The reading of the results is either by running a gel-electrophoresis or (B) visual inspection through the addition of SYBR Green with positive results presenting as a Subtyping & pathotyping of avian influenza viruses with green colour. Three positive samples are seen on the left and one a one-step real-time SYBR Green RT-PCR assay negative on the right, on both panels (A) and on (B). In addition, For the rapid subtyping and pathotyping of AIVs, a one-step (A) shows a size marker on the very left. real-time SYBR Green RT-PCR assay was developed. Primers were selected to target highly conserved nucleotide stretches that Real-time PCR assay based on primer-probe energy- flank the cleavage site of the HA gene of AIVs. Sequencing the transfer to detection swine vesicular disease virus amplified PCR products in AIV subtypes, including the H5 sub- A real-time PCR assay to detect swine vesicular disease virus types, allowed the rapid detection of the pathotype of AIV. The (SVDV) based on PriProET has been developed. The assay is specificity of the assay was confirmed by testing 27 strains of AIV highly sensitive with a detection limit corresponding to five copies and nine heterologous pathogens, including influenza B and C, of viral genome equivalents, and has a high specificity demon- and various other avian viruses. Since the subtype and pathotype strated by testing with heterologous viruses. A major advantage of determinations were completed within approximately 6 h, the the PriProET chemistry is a tolerance of mutations in the probe SYBR Green RT-PCR assay provides a powerful new tool in the region. Melting-curve ana lysis directly after PCR, with determi- arsenal of influenza diagnostics [52] . nation of probe melting point confirms the specific hybridization of SVDV strains. There is a high probability of identifying phy- Proximity ligation: protein detection by1 logenetically divergent strains of SVDV that may appear negative nucleic acid amplification in other probe-based real-time PCR assays. Moreover, any dif- Proximity ligation is based on the recently established proximity ference in melting points may provide an indication of mutation ligation mechanism that enables sensitive high-capacity protein divergence in the probe region [15] . detection, identification and measurement by converting detected specific proteins to an ana lysis of DNA sequences. Proximity liga- Simple & rapid detection of SVDV with a one-step tion enables a specific and quantitative transformation of proteins reverse transcriptase loop-mediated isothermal present in a sample into nucleic acid sequences. As pairs of so- amplification assay called proximity probes bind individual target protein molecules A one-step reverse transcriptase (RT)-LAMP assay was devel- at distinct sites, these reagents are brought in close proximity. oped recently to improve the detection of SVDV [40] . The assay These probes consist of a protein-specific binding part coupled provided a wide SVDV strain-detection range, since all 28 tested to an oligonucleotide with either a free 3´- or 5´-end capable of isolates of this virus were tested positive during the developmen- hybridizing to a common connector oligonucleotide. When the tal phase. Simultaneously, RT-LAMP yielded high specificity, as probes are in proximity, promoted by target binding, then the all tested heterologous viruses gave negative results for viruses DNA strands can be joined by enzymatic ligation. The nucleic such as FMDV and vesicular stomatitis. Since SVDV, FMDV acid sequence that is formed can then be amplified and quantita- and vesicular stomatitis virus all cause similar symptoms, the tively detected in a real-time monitored PCR. high specificity of detection and identification of SVDV is very This convenient assay is simple to perform and allows highly important in differentiating these diseases. RNA from clinical sensitive protein detection. Nordengrahn et al. reported that samples that included nasal swabs, serum and feces where used to detection sensitivities were similar to those for nucleic acid- evaluate the performance of the RT-LAMP with real-time PCR based detection reactions for the rapid detection of FMDV [53] . assays. When testing nasal swabs and serum, the sensitivity of the All indications are that the sensitivity of proximity ligation is assays were not significantly different, but, with fecal samples, the on a par with antigen ELISA (Belák s, thorén p, leBlanc n et al . pers. RT-LAMP assay performed significantly better than real-time oBserv.) and, therefore, could prove to play a significant role in PCR. The RT-LAMP assay has several features that supports its the early and rapid diagnosis of infectious diseases (including applicability as a powerful new tool of SVDV detection: biodefense). This platform is presently being adapted to be able to 8 Expert Rev. Mol. Diagn. 9(4), (2009)
  9. 9. Trends in viral diagnosis & epidemiology Reviewdetect the surface antigens of viruses (e.g.,AIV). The combination of nucleic acid and BVDV-1antigen detection approaches contribute toa complex, multilateral diagnosis of TADs. NADLIn addition, replacement of antigen ELISA Oslosswith a more sensitive and robust assay will BVDV-2be a significant advancement in the diag-nosis of infectious diseases. BVDV-3? 178003 890Magnetic bead-based microarray TKKfor detection & discriminationof pestivirusesA novel assay was developed for the rapid Hobidetection and discrimination of pestivi-ruses (i.e., BVDV types 1 and 2, CSFV and CSFVborder disease virus) using magnetic-beaddetection of PCR products in microarrays. AlfortAfter amplification, the PCR products are Giraffehybridized onto an array, followed by visu-alization with streptavidin-coated magnetic Bresciabeads for visual inspection or microscope BDVexamination, and this makes this novelassay suitable for use in modestly equipped Reindeerlaboratories. A panel of pestiviruses com-prising members of all the four accepted 137/4species was used to evaluate the assay with Moredunother post-PCR detection methods (e.g.,gel electrophoresis and suspension micro-array) used as comparisons to determinatesensitivity of the assay. The results clearly Figure 6. Molecular epidemiology, identification of new pestiviruses, includingdemonstrated that the assay provided a Th/04_KhonKaen. Unrooted phylogram generated from a fragment of the 5´NCRnovel, robust, sensitive and specific method sequences of selected representatives of each known species within the genus Pestivirus, including the tentative pestivirus of giraffe, the D32/00_‘HoBi’ and Th/04_KhonKaen.for the improved detection and discrimi- The sequences of pestivirus reference strains and previously described strains werenation of viral pathogens. In view of the obtained from the GenBank. The phylogram shows the phylogenetic position of thesimplicity of the assay and the very simple newly detected pestiviruses HoBi and TKK as new variants of BVDV (BVDV-3 perhaps).detection procedure in particular, this mag- For details see [55] .netic bead-based assay offers a powerfuland novel technology for molecular diagnostics in virology [54] . bovine viral diarrhea control. This spread also has implications for the biosafety of vaccines and other biological products, producedDetection of an emerging pestivirus by means of with fetal calf serum.molecular diagnostics & reverse genetics Virus isolation was not possible since the virus in the serumDuring a study of bovine viral diarrhea epidemiology in Thailand had been inactivated during processing; thus, reverse genet-using indirect antibody ELISA, an antigen ELISA and PCR a ics methods were applied to ‘reconstruct’ the inactivated viruspestivirus was detected in heat-inactivated serum of a calf. The until it regained its capacity to grow in cell cultures by usingPCR product was sequenced and comparative nucleotide sequence transfection of viral nucleic acids. Full-genome characterizationana lysis showed that this virus was closely related to a recently and phylogenetic ana lysis of the new virus were performed afterdescribed atypical pestivirus (D32/00 ‘HoBi’) that had been recuperation of the inactivated virus and, based on this, it wasfirst isolated from a batch of fetal calf serum collected in Brazil demonstrated that the virus was closely related to BVDV, sug-(Figure 6) . It was also demonstrated that the Thailand virus (known gesting that Th/04_KhonKaen and other HoBi-like pestivirusesas Th/04_KhonKaen) was circulating in the Thai herd [44] . The constitute a third genotype of BVDV (i.e., BVDV type 3) [55] .study was the first to report a natural infection in cattle with avirus from this group of atypical pestiviruses. The data suggest Microfluidic platforms & nanoscale reactionsthat these viruses had been spread to cattle populations in vari- Droplet-based microfluidics is a recent technology for high-ous regions of the world. If this global spread has occurred, then throughput parallel PCR analyses, which allows for millions ofatypical bovine pestiviruses will have important implications for discrete picoliter reactions [56] . There are commercial enterprises,www.expert-reviews.com 9
  10. 10. Review Belák, Thorén, LeBlanc & Viljoensuch as Raindance Technologies, which develop and drive such Characterization of new, hitherto unknown, viruses is oftenplatforms. The previously mentioned BioTrove is another exciting complicated by:technology, which offers high-throughput parallel analyses. These • Inability or very poor capacity to grow in cell culturestechnologies, with the amount of information they can provide,will find a place in both diagnostics and in molecular epidemiol- • Low copy number or unusual virion structure, limiting theogy sooner rather than later, as they bring huge advantages to the detection and identification of the virions with electron micros-early and rapid detection of disease pathogens (even before the copyonset of disease). • Being a minor part of mixed viral infections, where the co- infecting viruses are dominantNew approaches to molecular epidemiologyConsiderable progress was made in the development of various • Limited antigenic/serological crossreactivity, affecting thenucleic acid-detection methods and molecular epidemiology. detection by antigen–antibody assays, such as antigen-captureTraditionally, inferring molecular phylogeny was constructed by ELISA, immunofluorescence or immunohistochemistrydistance-based ana lysis (i.e., neighbor joining for single genes). In • The lack of nucleic acid identity or similarity to known virala recent study at the OIE Collaborating Centre for Biotechnology- sequences, thus, remaining undetected in nucleic acid hybrid-based Diagnosis of Infectious Diseases in Veterinary Medicine, a ization assaysBayesian ana lysis was carried out to analyze five genetic regionsof BVDV genome (5-UTR, Npro, E2a, E2b and NS3) for 68 taxa • Remaining undetected even by PCR assays, due to uniqueretrieved from GenBank. The best outcome for the ana lysis was genome structuresachieved when analyzing genetic regions with appropriate pro- In recent metagenomic studies, viral nucleic acids were detectedportions of conserved and variable sites or a combined dataset in uncultured environmental and clinical samples by using ran-consisting of all five genetic regions. In the future, Bayesian ana- dom amplification of their nucleic acids prior to subcloning andlysis combined with other traditional tree-building methods can sequencing. Already known and novel viruses were then identifiedbe used to estimate a more reliable viral phylogenetic tree and to by comparing their translated sequence to those of viral proteinsstudy the emerging and/or occurrence new variants of BVDV [57] . reported in public sequence databases. A wide range of specimens, such feces, serum, plasma, respiratory secretions and organ sus-Full-genome amplification of viral genomes pensions, seawater and near-shore sediments, was tested by viralTo systematically identify and analyze viral genomes (e.g., the metagenomic approach. Viral metagenomic approaches provide15 hemagglutinin and nine neuraminidase subtypes of influenza novel opportunities to generate an unbiased characterization ofA virus), reliable and simple methods that not only characterize the viral populations in various organisms and environments. Forpartial sequences but also analyze the entire genome are needed. example, such techniques have led to the detection of new parvo-It is possible to generate full-length cDNAs to subtype viruses, viruses, termed bocaviruses, in lower respiratory tract infectionssequence their DNA and construct expression plasmids for reverse of children [60] . By detecting a range of new viruses recently, viralgenetics systems by the selection and construction of specific sets metagenomics has broadened known viral diversity and openedof primers [58] . a very interesting new area in virological research and diagnosis By the use of new sequencing machines, such as the 454 sequenc- [61] . It is worthy to mention that the aforementioned approachesers (Roche, NJ, USA) [108] , approximately 30–60 million nucle- to determine viral diversity are not completely different and inde-otides can be sequenced within several hours (the number of pendent of each other as they are frequently used together in anucleotides is constantly increasing). The technique is extremely complex assay. For example, full-length amplification and full-important in the genetic research, including the investigation and length sequencing of viral genomes is a logical combination forcomparison of the viral genomes. This approach will lead to exact this approach to viral identification.information being obtained about the full-length sequences ofthe tested viral genomes and, therefore, they will provide more- Expert commentaryreliable data for determination of evolutionary aspects, relation- Considering the serious impact and socioeconomic effect ofships of viruses, viral subpopulations and many other important infectious diseases in human and in animal health today, thereaspects of molecular virology [59] . is a need for the very rapid development of novel, highly sensitive and robust biotechnology-based diagnostic techniques, whichUsing viral metagenomics to search for unknown viruses facilitate the early detection and identification of pathogens andSince, so far, unknown and/or recently emerging or re-emerging support the immediate implication of disease prevention andviruses are very important factors in the emergence of various control measures. The globalization of international trade, theinfectious diseases, it is important to work on methods that facili- rapid and free movement of large groups of people, animals,tate the detection of these unsuspected agents. In order to facili- food and feed products at a global scale has created a novel andtate the detection of such viruses, a range of molecular methods epidemiologically highly vulnerable situation. In this novel sce-have been developed to genetically characterize new viruses with- nario, both known infectious agents and/or newly emergingout prior in vitro replication or the use of virus-specific reagents. pathogens have the chance to spread all over the world within10 Expert Rev. Mol. Diagn. 9(4), (2009)
  11. 11. Trends in viral diagnosis & epidemiology Reviewseveral hours or days, causing serious epidemics. Regarding this diagnosis and provides powerful novel means for the elucidationepidemiological risk, it is extremely important to develop and of the infection biology of various diseases scenarios, such asapply powerful techniques, which are able to detect and identify respiratory or enteric disease complexes, where various virusesthe variants of the pathogens very rapidly and effectively. During and bacteria establish coinfections, influencing and/or aggravat-the past several years, numerous developments have taken place ing the effects of each other. The microarrays allow a very com-in the field, including a variety of advances in the real-time PCR plex investigation of these scenarios. The further developmentmethods, such as PriProET and LATE-PCR. The purpose is to of microarrays includes improvement of sensitivity (e.g., withincrease the diagnostic robustness of the PCR-based diagnosis combination of amplification processes). Specificity and detec-and to introduce various simple technical platforms, such as tion range is further improved by combination of wide-range andportable PCR machines, which allow the application of PCR highly specific target sequences. The infection-biology investiga-under field conditions for ‘point-in-care’ or on-site detection of tion is improved so that microarrays will detect not only severalthe pathogens. The introduction of novel isothermal amplifica- pathogens, but, simultaneously, they will measure the immunetion techniques, such as Invader and LAMP, is further facili- response capacities of the hosts (e.g., cytokine profiles), antibi-tating the adaptation of amplification-based techniques for the otic-resistance values, pathogenicity markers, population kineticsfront-line diagnosis of infectious diseases. The on-site detection and other important factors of disease prognosis, developmentof pathogens is further facilitated by other simple diagnostic and control. Microarrays, especially the liquid-phase approaches,tools, such as ‘dip-stick’ tests, including lateral-flow technique- will also allow easy automation and the use of high-throughput,based simple devices, which are able to detect and identify the robust diagnostic platforms.pathogens directly in the disease outbreaks, in human medical The arsenal of biotechnology-based diagnostic assays will beand in animal clinics, in animal farms and transport conditions rapidly expanded in the coming years by the routine diagnosticor in the food chain, among others. The introduction of the on- application of a variety of further methods, such as improvedsite diagnosis provides novel facilities and strong support to the detection of antigens by proximity ligation and by otherauthorities in prevention and control of infectious diseases, since approaches. The proper identification of virus variants and sub-the rapid, first-line diagnosis is extremely important. However, populations by full-genome sequencing will be affordable to manyit has to be considered and accepted that, in general, but espe- laboratories, since prices are dropping and facilities and servicescially in case of highly contagious and devastating infectious will be provided. The ana lysis of a large number of viral genomediseases, such as TADs, the on-site methods have to be confirmed sequences by powerful programes of bioinformatics will provideby laboratory-based diagnosis and pathogen identification. The novel tools for viral metagenomics, molecular epidemiology andlaboratory-based direct or indirect diagnosis is based on a wide disease control. The novel methods of indirect diagnosis, suchrange of confirmatory techniques of pathogen detection and as liquid-phase microarrays, completing or replacing ELISA,pathotype identification. will also facilitate the processes of pathogen detection and rapid To date, it is a positive tendency that a wide range of cen- identification, as well as the understanding of complex biologytral and field laboratories can afford the purchase and use of of infectious diseases.various real-time PCR machines. By the wider introduction of Since emerging and re-emerging pathogens, as well as ‘new’isothermal amplification assays, the equipment facilities of the viruses, are posing a serious threat to animal and human health,laboratories are further improved, considering that this diag- including problems of zoonoses, it is very important to developnosis requires only simple low-cost thermoblocks. Since sample and apply powerful approaches for new virus detection and identi-preparation, such as nucleic acid extraction, is another important fication. In the last few years, a range of such techniques have beenstep of molecular diagnosis, many laboratories purchase nucleic developed. So far, these methods were able to detect mainly smallacid extraction and pipetting robots. By robotization the nucleic DNA viruses, such as bocaviruses and transfusion-transmittedacid-based diagnosis is saving manpower, and the automated virus. However, there is a need to further develop the new virus-processes allow high-throughput diagnosis. Simultaneously, the detection techniques, with special regard to the detection of vari-crosscontamination and carryover risks, which were considered ous RNA viruses, which play an important role in TADs, humanas Achilles heels of PCR diagnosis for a decade, are minimized. epidemics and emerging new diseases worldwide.If we recall how much the introduction of the automated ELISA Importantly, diagnostic laboratories should be encouraged todiagnostic systems did improve the reliability and speed of diag- work simultaneously in two directions to introduce the novelnostic work in the last 20 years, then we can state that a similar biotechnology-based diagnostic techniques, as outlined in thisprocess in the PCR-based diagnosis is extremely important and article to maintain the knowledge of classical virology and bac-useful today. teriology, with special regards to virus isolation in cell culture, In parallel with the PCR-based diagnosis, a high range of other and in vitro and in vivo characterization of viruses and bacteria.diagnostic techniques have been introduced in the diagnostic Although classical virology and bacteriology are not the subjectlaboratories, as it is outlined in this review shortly. We would like of our present article, we feel compelled to mention this hereto emphasize the use of solid- or liquid-microarray techniques, because we see that this profile of the diagnostic laboratories iswhich allow the simultaneous detection of a very wide range decreasing dramatically. This is a very dangerous tendency, con-of pathogens in the same platform. This allows a very complex sidering that we cannot control infectious diseases effectively ifwww.expert-reviews.com 11
  12. 12. Review Belák, Thorén, LeBlanc & Viljoenwe loose the capacity to isolate and cultivate virus strains, which increasing, very important requirement today, at a global scale.are absolutely important to understand the pathogenesis of the Considering this requirement, research will be conducted to detectagents to develop diagnostics and vaccines and for many other and identify the various infectious agents, chemicals, hormonespurposes of infection biology. and other factors of food safety, in very robust and complex diag- Considering these facts, we believe that a practical combination nostic systems. Simultaneously, research will focus on questionsof classical and biotechnology-based microbiology is required to of zoonotic infections including the transmission of infectionscombat infectious diseases effectively today and in the future. between various hosts in the animal and human populations. The climatic variations and changes, observed worldwide, raiseFive-year view a number of questions for the emergence of new pathogens andThe detection and characterization of specific nucleic acids of the spread of diseases to new geographic areas, where they weremedicoveterinary pathogens have proven invaluable for diagnostic not previously known. Since the geographic territories and thepurposes. Apart from hybridization and sequencing techniques, population biology conditions of insect vectors are strongly influ-PCR and numerous other methods have contributed significantly enced by climatic changes, the issue of emerging vector-borneto this process. The integration of amplification and signal-detec- diseases, such as bluetongue, has become a very hot issue in recenttion systems, including online real-time devices, have increased years. It is evident that this tendency will be clearly seen, even inspeed and sensitivity and greatly facilitated the quantification of the coming period of time. The appearance of ‘so far unknown’target nucleic acids. They have also allowed for sequence charac- or ‘completely new’ pathogens will create new epidemiologicalterization using melting or hybridization curves. Rugged portable situations in large regions of the world.real-time instruments for field use and robotic devices for process- The ‘one world, one health’ principle is extremely important,ing samples are already available commercially. Various stem-loop and it will be even more important in the future. Our world ofDNA probes have been designed to have greater specificity for extremely intensified globalization, international trade and globaltarget recognition during real-time PCR. DNA fingerprinting tourism, struck by climatic changes, financial, energy and politicaltechniques or postamplification sequencing is used to type patho- turbulences, has become extremely sensitive and vulnerable con-genic strains. Characterization according to DNA sequence is cerning the subjects of this article: transboundary and endemicbecoming more readily available as automated sequencers become diseases, food safety and zoonoses.more widely used. Reverse hybridization and, to a greater degree, If we try to outline a 5-year view, it is clear that there will be aDNA microarrays (as an example of high-throughput systems), are vast need for the rapid, robust, high-throughput complex detec-being used for genotyping related organisms, and can allow for the tion of known and unknown pathogens. The tendency of ferventdetection of a large variety of different pathogens simultaneously. development is clearly indicated in the listed examples of thisNonradioactive labeling of DNA, especially using fluorophores article, which provides a short review of recent technologies thatand the principles of fluorescence resonance energy transfer, is now we believe will transform certain areas in molecular diagnosticwidely used, especially in real-time detection devices. virology. This field will continue to develop rapidly in the com- Other detection methods include the use of surface plasmon ing years, with many novel approaches for virus detection andresonance and matrix-assisted laser desorption ionization time- characterization. Highly specific single-pathogen identificationof-flight (MALDI-TOF) mass spectrometry. In addition to these assays and very complex, wide-pathogen-range, high-throughputtechnological advances, contributions by bioinformatics and the diagnostic panels will be simultaneously developed and applieddescription of unique signatures of DNA sequences from patho- for prompt detection and identification of pathogens.gens will contribute to the development of further assays for The diagnoses will be extended to complex questions ofmonitoring presence of pathogens. An important goal will be the ‘infection biology’, where not only ‘known pathogens’ will becontinuous development of robust devices capable of sensitively detected in complex diagnostic panels, but also ‘so far unknownand specifically detecting a broad spectrum of pathogens that will agents’, which may influence the health conditions of animalsbe applicable for point-of-care use. Advances in biosensors, the and humans by themselves, or in synergetic effects with otherdevelopment of integrated systems, such as lab-on-a-chip devices infectious agents, creating a very complex scenario of infectionand enhanced communications systems, are likely to play sig- biology. Panels, such as wide microarrays, will be developed,nificant roles in allowing for rapid therapeutic and management which detect not only infectious agents, but investigate theirstrategies to deal with disease outbreaks. populations and subpopulations, evolutionary aspects, in con- It is evident that the molecular diagnosis of infectious trans- nection to the immune defence capacities of the hosts, such asboundary and endemic diseases of animals and man will show cytokine expressions, or genetic, nutritional and other factors ofa great deal of progress in the coming years. Intensive research complex disease development. Special attention will be paid towill also be conducted in the field of food safety, considering the the development of complex diagnostic panels to detect emerg-need for safe food products in our world of intensive globaliza- ing and re-emerging diseases, as well as maladies connected totion, where food products are transported at continental or at a climatic changes. Simultaneously, research will be conducted onglobal scale, causing problems for identification of the origin of the effects of agents, which are probably not direct pathogens bythese products and ensuring food safety requirements. The safe themselves but, in combination with other factors, are certainlytracing, identification and testing of food and feed products is an capable of initiating or aggravating complex disease scenarios.12 Expert Rev. Mol. Diagn. 9(4), (2009)
  13. 13. Trends in viral diagnosis & epidemiology Review The wide range of methods of pathogen detection and identifi- István Kiss (SVA), critical reading. Thanks also to Cor Schoen (PRI-WUR,cation outlined in this article, serves as a stable basis for method Netherlands) for providing the photographs for Figures 1& 3, as well as technicaldevelopment in the coming years. A wide range of additional meth- information on the BioTrove OpenArray system. We also gratefully acknowl-ods of direct and indirect virus detection will be developed, based edge the contribution of our partners and funding agencies in the LAB-ON-on further technological approaches, such as nanotechnology and SITE project of the EC (SSPECT-2004-513645), EPIZONE Network ofother powerful technical principles. By using all these novel tech- Excellence (FP6-2004-Food-3-A-016236), DG Research of the EC, the OIEnologies to address the listed wide range of problems, there is a and SLU “Excellensbidrag”.hope that the one world, one health principle will be followed andthe quality of human and animal life will be improved. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with anyAcknowledgements organization or entity with a financial interest in or financial conflict withThe authors would like to thank all their colleagues and, in particular, for the subject matter or materials discussed in the manuscript. This includestheir critical reading, figures and photographs: Anne-Lie Blomström (SLU, employment, consultancies, honoraria, stock ownership or options, expertSweden), Figure 5 ; Lihong Liu (SLU), Figure 6 ; Mikhayil Hakhverdyan (SVA, testimony, grants or patents received or pending, or royalties.Sweden), Figure 2 ; Carmelo Volpe (Smiths Detection, UK), Figure 4 ; and No writing assistance was utilized in the production of this manuscript.Key issues• The early and rapid diagnosis and control of disease pathogens – preferably before the onset of disease, or an outbreak.• Real-time confirmation of disease suspicion between field and laboratory.• Transfer of appropriate and sustainable technologies.• Real-time implementation of control measures based on the timely diagnosis and confirmation of diagnosis.• Harmonization and quality control of molecular diagnostic assays to ensure that tests used are fit for their intended purpose and that results produced are globally comparable.• Portable technologies, both simple and sophisticated, are poised to make an impact on the diagnosis of viral diseases as well as the way in which epidemiological data can be obtained.• New technologies, such as microfluidic-based PCR and rapid sequencing, will provide massive quantities of data. One challenge will be the managing and interpretation of this information.• The present advances in technologies are in amplification technologies, such as PCR, to achieve higher sensitivity and specificity.• Future advances will be in direct detection technologies – the detection of a virus on molecular level without the need to amplify its genetic material.References 7 Belák S, Thorén P. Molecular diagnosis of Viral Diseases in Veterinary Medicine”.Papers of special note have been highlighted as: animal diseases: some experiences over the Dtsch Tierärztl. Wochenschr. 113, 129–133• of interest past decade. Expert Rev. Mol. Diagn. 1, (2006). 434–443 (2001). 12 McKillen J, Hjertner B, Millar A et al.1 Lubroth J, Rweyemamu MM, Viljoen GJ et al. Veterinary vaccines and their use in 8 Belák S. The molecular diagnosis of Molecular beacon real-time PCR detection developing countries. Rev. Sci. Tech. 26, porcine viral diseases: a review. Acta Vet. of swine viruses. J. Virol. 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