key: cord-0034821-uhei3bvr
authors: Belák, Sándor; Liu, Lihong
title: Recent Advances in Veterinary Diagnostic Virology: Report from a Collaborating Centre of the World Organization for Animal Health (OIE)
date: 2012-04-05
journal: Advanced Techniques in Diagnostic Microbiology
DOI: 10.1007/978-1-4614-3970-7_36
sha: 1933570de05de8d0f1fa9d58993c2e206bb4095f
doc_id: 34821
cord_uid: uhei3bvr

Infectious diseases have a very high impact on animal and human health and welfare today, despite of strong efforts and good results in diagnostics, vaccine developments and control measures, including the early warning systems. There are many reasons, which have to be considered as supporting factors for the spread of infectious diseases, such as the open borders of the European Union, allowing rather free movement of animals over a whole continent, the globalization, the released and accelerated international and national trade and animal transfer. Simultaneously, the emergence and re-emergence of new or already known pathogens is a various serious issue in veterinary and in human medicine. This scenario is clearly illustrated by the regular occurrence of transboundary animal diseases (TADs), such as foot-and-mouth disease (FMD), classical swine fever (CSF), African swine fever (ASF), among others. The recent occurrence of African swine fever in the Caucasus region and the spread afterwards to large territories of Russia clearly illustrates that our health authorities require a very strong preparedness, including prompt and powerful diagnosis, for the successful fight against the novel scenarios.

very strong preparedness, including prompt and powerful diagnosis, for the successful fi ght against the novel scenarios.

Considering these requirements the European Union (EU) is strongly supporting the development of powerful novel diagnostic methods, which are assured by international comparison, standardization and validation. Thus, the EU has launched and supported a large range of various projects for these purposes recently.

At the World Organization for Animal Health (OIE) Collaborating Centre for the Biotechnology-Based Diagnosis of Infectious Diseases in Veterinary Medicine , at the National Veterinary Institute (SVA) and the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden, we have participated/participate in a high number of these projects, such as LAB-ON-SITE, EPIZONE, FLUTRAIN, FLUTEST, CSFV_goDIVA, ASFRISK, ConFluTech, AsemDialog, among others. As an example, the LAB-ON-SITE project (coordinated by SB) strongly contributed to the development of veterinary diagnostic virology. Ten infectious TADs, listed as noti fi able to the OIE-FMD, swine vesicular disease (SVD), vesicular stomatitis (VS), CSF, ASF, bluetongue (BT), African horse sickness (AHS), Newcastle disease (ND), highly pathogenic avian in fl uenza (HPAI) and swine in fl uenza (SI)-were the subjects of this international research project of the EU. A range of new diagnostic assays were developed to to improve the current detection of the targeted ten viruses, such as novel real-time PCR assays, isothermal ampli fi cation methods, padlock probes, and novel ELISA systems. In order to improve the front-line, on-site diagnosis in the fi eld, simple methods and equipment were adapted, such as portable PCR machines, simple thermo-platforms for isothermal ampli fi cation, dipsticks and lateral fl ow devices. Many of the developed methods were compared in the large international consortium, validation was performed and standard operating procedures (SOPs) of the new methods were disseminated via international organizations, such as the OIE with its 178 member countries and the International Atomic Energy Agency (IAEA) with its large networks in Asia, Africa and South America. Honouring the strong output, this project was considered as one of the Success Stories in FP6 of the European Union ( http://ec.europa.eu/research/agriculture/success_labonsite_en.htm ).

Considering the above-listed scenarios and requirements, the prompt detection and very rapid and exact identi fi cation of various pathogens is a very important and essential task in veterinary virology. While classical diagnostic methods, such as virus isolation, remain technically unaltered or show rather little steps of changes and development, the molecular diagnostic methods have advanced dramatically in the last decades. These new techniques provide powerful novel tools for the rapid detection and identi fi cation of a wide range of causative agents, as well as for supporting disease control and surveillance. For example, real-time quantitative PCR assays are highly sensitive and speci fi c methods, which are widely used for detection of various pathogens in diagnostic units, including the Reference Laboratories and the Collaborating Centres of the World Health Organization (WHO; http://www.who.int ) and of the World Organization for Animal Health (OIE; www.oie.int ).

At the OIE Collaborating Centre for the Biotechnology-Based Diagnosis of Infectious Diseases in Veterinary Medicine , Uppsala, Sweden, great efforts have been made for the development, standardization and dissemination of new diagnostic assays within the frame of several large EU and other projects in the recent years. Some achievements have been summarized in previously published review articles [ 1, 2 ] . Herewith, an update and selected examples are given in order to illustrate the trends of recent advances in veterinary diagnostic virology at our OIE Collaborating Centre, in collaboration with a wide network of partner laboratories in Europe and on other continents.

Proper sample collection, storage, transportation and enrichment are crucial for the reliable diagnosis of infectious diseases. To avoid potential degradation of the targeted nucleic acids, the samples are normally transported at low temperature using dry ice, cooling batteries and/or are stored in −20/70 °C freezers before further processing. Heat-and/or chemical inactivation of samples is required to avoid the transmission and spread of infectious agents. In order to diagnose the various known and "unknown" diseases in a safe and reliable way, proper methodologies are needed, which simplify the handling, processing of the samples, and the transport to the diagnostic laboratories. Currently, several simple tools, such as different fi lter papers or cards are commercially available for such purposes. For example, FTA™ Cards, produced by Whatman plc, now a part of GE Healthcare, are suitable for the collection, transportation and storage of samples at room temperature. The company claimed that such FTA Cards contain chemicals that lyse cells, denature proteins and protect nucleic acids from nucleases, as well as from the damages caused by heat, oxidative and UV effects. At the Collaborating Centre, our colleagues have used these types of cards for transportation of samples from other countries and continents to Sweden. For example, ASF serum samples were collected in Uganda, placed on the fi lter papers, and transported to Sweden at room temperature. The same type of fi lter papers was used for transportation of Peste des Petits Ruminants (PPR) samples from Pakistan. Bat coronavirus samples were sent from Brazil to Sweden in an envelope, by post. Viral nucleic acids, eluted from the cards, are successfully ampli fi ed by various PCR assays or detected by other means of molecular diagnostic virology. We perform real-time PCR assays, using small pieces of cards containing samples as templates and various viruses are detected in a convincing way after this simple way of sample transportation.

The diagnosis in veterinary virology is frequently complicated by the fact that the amount of targeted viruses in the certain types of clinical samples, in food and feed products or in water samples, as well as in other diagnostic specimens, is often very low. The low target content may lead to false negative results in the various PCR assays and in other molecular diagnostic procedures. In order to avoid or to reduce this very important bottleneck effect, diagnostic laboratories develop and apply a range of sample enrichment methods, in order to " fi sh out" the targeted pathogens or their components, such as nucleic acids or proteins, from the analyzed specimens. For example, DNA enrichment is achieved by functionalized magnetic nanoparticles that are coupled with probes to which the target DNA could be hybridized [ 3 ] .

The proper preparation of viral nucleic acids as targets of PCR and other molecular diagnostic assays is also a very important task in the diagnostic laboratories. Simplicity and high-throughput capacity are major concerns in case of outbreaks where a huge number of samples are processed within a relatively short period of time. It is almost impossible to complete such a task by manual extraction methods such as TRIzol reagent or spin columns. The manual extraction processes are labourand time-consuming and expensive. In addition, personnel costs and working processes have to be considered. For those reasons, various kinds of automated equipment have been developed and commercialized for nucleic acid preparation and/or handling of samples. At our OIE Collaborating Centre the automated nucleic acid extraction steps are performed with robotic extraction machines.

In an exercise to compare the performance of nucleic acid extraction robots (12 separate instruments, comprising eight different models) in fi ve European veterinary laboratories, similar results were observed from best performing robots when dilutions of a cell culture supernatant were tested, whereas up to 1,000-fold difference was obtained from less optimized robots when dilutions of a serum sample were tested [ 4 ] . It was observed that the same instrument performed differently when tested with different types of samples. Therefore, a pro fi ciency test or "ring trial" of different clinical samples would be of great help to validate in-house assays, identify critical steps and improve performance of downstream PCR tests.

Since the publication of quantitative real-time PCR in 1996 [ 5 ] , it has become a very reliable, powerful, high-throughput and robust molecular tool for early, rapid and sensitive detection of pathogens in both human and veterinary medicine. Compared to the conventional gel-based PCR, the real-time PCR has several advantages, including high-throughput capacity, less hands-on time, lower risk of contamination, and the potential to be fully automated [ 6 ] . By using a gene-speci fi c probe, the speci fi city and analytic sensitivity of the real-time PCR may be further improved. The research activities at our Collaborating Centre aimed to develop real-time PCR assays for diagnostics to differentiate infected from vaccinated animals (DIVA), for speci fi c detection of emerging viruses, and for detection of zoonotic agents, in particular water-and food borne pathogens.

Herewith, several examples are presented regarding speci fi c problems, illustrating the diagnostic application of various real-time quantitative PCR assays developed and used at our OIE Collaborating Centre, in collaboration with other European veterinary institutes in various research projects of the European Union. Further important achievements are also listed here, for example the development of real-time RT-PCR assay using a minor-groove binder probe for the pan-serotypic detection of foot and mouth disease virus (FMDV) [ 7 ] and the application of loop-mediated isothermal ampli fi cation (LAMP) for detection of wild type classical swine fever virus (CSFV) [ 8 ] and vaccine strain [ 9 ] , and swine vesicular disease virus (SVDV) [ 10 ] .

CSFV is a pestivirus within the family of Flaviviridae . It is the causative agent of classical swine fever (CSF), a highly contagious disease affecting both wild boars and domestic pigs. In Europe, the virus is largely maintained in the wild boar populations that serve as a reservoir for reintroduction to domestic pigs. Recently, a chimeric vaccine candidate, CP7_E2alf has been developed and has the potential to be used as a safe and ef fi cient marker vaccine in wild boars, which enables differentiation of infected from vaccinated animals [ 11, 12 ] . A vaccine-speci fi c TaqMan real-time RT-PCR assay was developed and evaluated, and a second, wild type-speci fi c assay was modi fi ed from an established one in such a way that both can be performed in two wells side-by-side in a microplate in a single run [ 13 ] . Both assays could be applied in CSFV vaccination and control programs in the wild boar population.

CSF remains endemic in domestic pigs in some countries, for example, China, where vaccination with the traditional lapinized live virus is still in practice. DIVA diagnostics is a potentially powerful tool to discriminate whether pigs are naturally infected with wild type strains or vaccinated with the lapinized live virus. The Chinese Hog Cholera Lapinized virus (HCLV) has been through extensive serial passages in rabbits in China in 1950s and is completely attenuated, but retains its ef fi cacy as vaccine. Due to its safety and ef fi cacy, this vaccine was introduced into European countries and named as "Chinese" strain (C-strain). "Riems" C-strain is one of the commercial vaccines derived from the Chinese strain. Development of a vaccine-speci fi c real-time RT-PCR would be of great value, which could be used in parallel with a wild type-speci fi c or generic real-time RT-PCR for detection and differentiation of wild type viruses from vaccine strains. Leifer et al. studied differentiation of Riems C-strain-vaccinated pigs from animals infected by CSFV fi eld strains using real-time TaqMan RT-PCR [ 14 ] . Leifer et al. also reported escape of CSF C-strain vaccine virus from detection by C-strain speci fi c real-time RT-PCR caused by a point mutation in the primer-binding site [ 15 ] . At the Collaborating Centre, we have developed a real-time PCR based on primer-probe energy transfer technology (PriProET) for the improved detection of CSFV [ 16 ] . The PriProET technology was developed initially as a novel quantitative real-time PCR assay for the simultaneous detection of all serotypes of FMDV [ 17 ] , and subsequently was used for detection of other pathogens, including SVDV [ 18 ] , and bluetongue virus (BTV) [ 19 ] . Following PCR ampli fi cation, the melting curve analysis allows con fi rmation of speci fi c amplicons, and differentiation between wild type CSFV and the HCLV vaccine strain (Fig. 36.1 ). Further evaluation of the assay demonstrated that in an RNA mixture of both wild type CSFV and C-strain vaccine, the melting curves displayed only one curve either a wild type-like or a vaccine-like depending on the dominating RNA [ 20 ] . Therefore, the PriProET melting curve analysis could identify the presence of CSFV fi eld strain in equivocal samples or in animals vaccinated with C-strain, but would not reliably detect infections with wild-type virus in a population vaccinated with the Riems strain. In a recent study, a generic real-time TaqMan RT-PCR was developed for the speci fi c detection of three lapinized vaccine strains, namely, the Taiwanese Lapinized Philippines Coronel (LPC), the Chinese HCLV, and the European Riems C-strain [ 21 ] . The new assay was able to detect the Riemser C-strain vaccine viral RNA in experimental samples, indicating that the assay could be a useful tool to facilitate outbreak control if a strategy using these lapinized vaccines is deployed.

Over the past few years, new bovine pestiviruses have been detected in biological products, e.g., foetal bovine serum (FBS) batches, and in naturally infected cattle. Due to nucleotide sequence variations, the identi fi cation of these viruses might fail, since primers and probes had mismatches to their target, leading to a false negative result. For example, the well-known "pan-pestivirus" primer pair 324/326 failed to react with D32/00_'HoBi' virus that was found in a batch of contaminated foetal calf serum [ 22 ] . To overcome the limitations of current molecular methods, a new one-step real-time TaqMan RT-PCR assay was developed for the speci fi c detection of atypical bovine pestiviruses in clinical samples and in biological products [ 23 ] . This new assay provides a useful tool for highly sensitive and speci fi c detection of atypical bovine pestiviruses and can be applied in combination with other diagnostic methods to ensure that biological products, including FBS and vaccines, are free from pestivirus contamination. Recently, we have tested 33 batches of commercial Tests showed presence of both the recognized species and the newly described atypical bovine pestivirus in the FBS batch. The contaminating atypical pestivirus was further characterized, as it is described by our group [ 24 ] .

Apart from pathogens only affecting animals, a high number of pathogens can cross the species barrier(s) and cause diseases in humans, which is referred as zoonosis.

Of more than 1,400 human pathogens, approximately 60 % are zoonotic, of which 25 % are estimated being able to be transmitted from human to human [ 25 ] . Therefore, it is of great importance to develop broadly targeting real-time RT-PCR for the generic detection of zoonotic viruses at our Collaborating Centre. Herewith, several examples are described.

Coronaviruses are the causative agents of a broad spectrum of animal and human diseases, including the Severe Acute Respiratory Syndrome (SARS) in humans. Considering this scenario, it is very important to develop very "general coronavirus" PCR assays, which are able to detect a wide range of viruses in various hosts simultaneously. In order to ful fi l these requirements, we have developed a pan-coronavirus real-time RT-PCR, which is able to detect 36 strains of animal and human coronaviruses simultaneously, including HCoV-NL63 and SARS-CoV [ 26 ] . In this PCR panel the degenerated primers are targeting a conserved region of the ORF1b of the viral and have a low annealing temperature. We have compared this real-time RT-PCR system with two published TaqMan assays for detection of four animal coronaviruses in 75 clinical samples, and obtained the same results.

It has to be mentioned that eventhough this SYBR Green-based PCR assay is very promising in the general detection of coronaviruses, it also has weakness with regards to its speci fi city. Apart from the coronavirus positive samples, primer dimers and non-speci fi c amplicons may also produce ampli fi cation curves and this can lead to false positive results. In order to avoid false diagnosis, melting curve analysis is needed in this assay. The melting curve analysis is a practical tool to verify the truly positive results. In summary, it was concluded that this SYBR Green-based realtime pan-coronavirus PCR assay, along with melting curve analysis, provides a powerful novel tool for the laboratory diagnosis of coronavirus infections and for detecting the emerging, yet to be uncharacterized new coronavirus variants. Considering the strong emergence rate of many RNA viruses, including the coronaviruses, the use of such pan-virus family PCR assays is very important.

The new assay provides a novel tool for our diagnostic laboratories in veterinary and in human virology.

Concerning other real-time PCR approaches, such as the TaqMan principle, we have found that it is challenging and dif fi cult to develop a pan-coronavirus detecting system using a single probe. Even though the genomic stretches are relatively conserved, it is almost impossible to design a TaqMan probe for ef fi cient general detection of coronaviruses. To overcome this limitation, three overlapping TaqMan probes were designed and used together with the above-mentioned primers for sensitive and speci fi c detection of coronaviruses of all three groups [ 27 ] . We have found this three-probe approach reduced the degree of probe degeneration and maintained a high sensitivity and speci fi city. The assay detected coronaviruses in human nasopharyngeal aspirate samples and in duck faecal samples. Furthermore, this method was able to detect SARS-CoV in a reference material, and detect the synthetic sequences of bat coronaviruses (HKU3-CoV, HKU5-Cov and HKU9-CoV), indicating it is a useful tool for screening samples of animal origin for zoonotic or potentially zoonotic pathogens. The assay was used as a tool for epidemiological studies on coronaviruses in wild birds from the Bering Strait area [ 28 ] .

Food-and waterborne zoonotic pathogens frequently cause large outbreaks in regions where sanitation is poor. Hepatitis E virus (HEV) is an important food-and waterborne zoonotic pathogen, which is an emerging virus of global importance. This single-stranded, positive-sense RNA virus belongs to the genus Hepevirus in the family of Hepeviridae that includes at least four recognized genotypes. While genotypes 1 and 2 are associated with large outbreaks and epidemics in humans in developing countries with poor sanitary conditions, genotypes 3 and 4 HEV strains are able to infect both humans and various animal species, in particular, domestic pigs and wild boars, and are responsible for sporadic cases of acute hepatitis E not only in the developing, but also in the industrialized countries. It has been recognized that hepatitis E is a zoonotic disease. At our OIE Collaborating Centre, Gyarmati et al. developed two real-time PCR assays for universal detection of all four genotypes of HEV [ 29 ] . Both the TaqMan and PriProET assays were able to detect 20 viral genome equivalents per reaction. The results obtained from both realtime PCR assays were comparable to those from a nested gel-based PCR. However, the TaqMan assay had higher reaction ef fi ciency and performed slightly better than the PriProET assay. Such tools are important not only for veterinary diagnostics but also for molecular epidemiology of HEV in humans, pigs and wild boars [ 30 ] .

It is not widely known yet that real-time PCR assays can be used not only for the detection of viral nucleic acids, but also for the detection of protein components or antigens of pathogens . This technique is referred to proximity ligation assay (PLA) [ 31 ] . In a proximity ligation assay, two oligonucleotides are coupled to antibodies. Binding of the antibodies to the same target will bring the two oligonucleotides suf fi ciently close to each other (or in proximity) such that the two ends could be ligated with the help of a third connector oligonucleotide. The ligation products are then used as target DNA for subsequent ampli fi cation and detection by real-time PCR. At the OIE Collaborating Centre in Uppsala, Schlingemann et al. used this technique to detect antigens of avian in fl uenza viruses in biological samples [ 32 ] . We have found that the method is four orders of magnitude more sensitive than a sandwich ELISA, which utilized the same antibody. This study opens a new window for diagnostic virology, by using PLA for the highly effective detection of viral proteins in clinical specimens.

Although real-time RT-PCR offers multiplex detection of pathogens, its capacity is limited to no more than approximately fi ve targets in a single diagnostic block. By contrast, suspension microarray technology, such as Luminex xMAP technology (Luminex Corporation, Austin, TX) has the capacity of detecting up to 100 targets in a single platform, or even more . At our OIE Collaborating Centre a range of Luminex assays have been developed for the multiplex detection of pathogens important in human and veterinary medicine. A suspension microarray assay was constructed for detection and differentiation of pestiviruses including CSFV, BVDV-1, BVDV-2, border disease virus (BDV) and the atypical bovine pestivirus [ 33 ] . Following PCR ampli fi cation of target genetic region, one strand of the amplicons was removed by Lambda exonuclease to generate single-stranded PCR products for ef fi cient hybridization to the microsphere-coupled oligo probes. After incubation with a reporter dye streptavidin-R-phycoerythrin, the median fl uorescent intensity (MFI) of each reaction was measured in a Luminex instrument. The system was able to provide positive/negative results and to identify each species by comparing MFI values. Such an assay is a useful tool for the genetic detection of various pestiviruses in cattle.

Another Luminex suspension microarray assay was developed at our laboratories for the serological diagnosis of bovine viral diarrhoea (BVD) [ 34 ] . In this blocking system, microspheres were coupled with a monoclonal antibody (MAb) and were incubated with viral antigen and serum samples. After addition of another MAb that is biotinylated, R-phycoerythrin-conjugated streptavidin was added and the MFI values were measured on a Luminex 200 analyzer. This blocking microsphere-based immunoassay had intra-and inter-assay variability of 4.9 % and <7 %, respectively, and variability of bead conjugations of <6.6 %. By testing a total of 509 serum samples, the assay had a sensitivity of 99.4 % and a speci fi city of 98.3 % relative to a commercial blocking ELISA. The new microsphere immunoassay provides an alternative to conventional ELISA systems and can be used for high-throughput screening in the BVD control and eradication program.

As BVDV is one of the pathogens causing bovine respiratory disease (BRD) complex, our ongoing work at the Collaborating Centre is to develop a multiplex Luminex assay for the high-throughput, simultaneous detection of antibodies against four major viruses that cause BRD, namely, BVDV, bovine respiratory syncytial virus (BRSV), parain fl uenza type-3 virus (PIV3) and bovine coronavirus (BCoV). This novel multiplex assay not only allows a higher sample throughput but also reduces the time and labour required. The Luminex platform has a strong potential to replace ELISA as a powerful novel system for the complex diagnosis of pathogens.

A pathogen's genome can be determined by various methods, such as the traditional cycle sequencing and by the advanced "next-generation sequencing" technologies. While the cycle sequencing is still the method of choice in most laboratories, next-generation sequencing is becoming available in some laboratories. Both methods are a powerful tool in veterinary diagnostic virology. Furthermore, genome sequencing will contribute substantially to a better understanding of pathogens, which would be helpful in veterinary diagnostic virology. Particularly, viral metagenomics is a generic technology using large-scale sequencing to identify viral genome sequences without prior knowledge. Viral metagenomics has helped researchers with the investigation of complex diseases, diseases of unknown aetiology, and identi fi cation of emerging novel viruses in samples. In the followings, several examples are given on the use of viral metagenomics at our OIE Collaborating Centre to detect new and/or emerging and re-emerging viruses in animals.

PMWS is a viral disease worldwide affecting postweaning pigs of about 2-4 months of age. Porcine circovirus type 2 (PCV-2) has been found to play an important role in the complex infection biology of PMWS. PCV-2 is a small circular DNA virus belonging to the family Circoviridae. The virus is ubiquitously distributed in almost all pig populations, not only in PMWS pigs but also in clinically healthy ones. Therefore, it is hypothesized that other known or unknown factors may have a contribution to the development of PMWS. Aiming to fi nd possible factors contributed to PMWS in piglets, our group tested lymph node samples that were collected from Swedish pigs with con fi rmed PMWS, by using GS-FLX 454 technology for large-scale sequencing of randomly ampli fi ed products [ 35 ] . Analysis of about 9,000 unique sequences obtained from the 454 run showed presence of PCV-2, Torque Teno virus genotypes 1 and 2 (TTV-1 and TTV-2), as well as a novel parvovirus-like agent, termed as porcine boca-like virus in the lymph nodes from the diseased pigs. Based on this study, Blomström et al. screened 34 PMWS pigs and 24 animals without PMWS by PCR for the presence of PCV-2, TTV-1, TTV-2 and porcine boca-like virus [ 36 ] . PCV-2 was found in all PMWS pigs and in 80 % of pigs without PMWS. Presence of all three viruses TTV-1, TTV-2 and porcine bocalike virus was found in 71 % of the PMWS pigs and in 33 % pigs without PMWS. The exact role of these viruses, particularly the porcine boca-like virus in the development of PMWS requires further investigations.

At our OIE Collaborating Centre viral metagenomic approaches, including the GS-FLX 454 technology was also used to investigate the cause of a neurological disease of minks termed shaking mink syndrome . The disease was fi rst observed in farmed mink kits in Denmark in 2000 and subsequently in Sweden, Denmark and Finland in 2001, and in Denmark again in 2002 [ 37 ] . General virological investigations, including virus culture, negative-staining electron microscopy, immunoelectron microscopy, broad targeting PCR for herpesviruses, adenoviruses, pestiviruses and coronaviruses, and speci fi c tests for six viral diseases, for three protozoa, for bacteria and intracerebral inoculation of neonatal mice were conducted but with negative results. It was postulated that it is likely that the disease was caused by a yet unidenti fi ed virus [ 37 ] . To investigate the cause, brain samples from experimental infection were prepared for nucleic acid extraction and random ampli fi cation and large-scale sequencing using GS-FLX 454 technology [ 38 ] . Analysis of the 454 sequencing data revealed eight sequence fragments similar to mink astrovirus. Based on the result, new primers were designed in order to determine the nucleotide sequences of the complete viral genome. The comparative analysis of complete genome sequences showed a similarity of 80.4 % to that of a mink astrovirus causing pre-weaning diarrhoea in mink. As the virus was not detected in healthy mink kits, we suppose an association between the astrovirus and the neurological disease of mink.

Genome sequencing and subsequent phylogenetic analysis have been considered as important tools for the exact identi fi cation of the "unknown" or emerging new pathogens. From the initial discovery of the pestivirus D32/00_'HoBi' in a batch of contaminated FBS, it was unclear how this virus is related to the two recognized bovine pestivirus species: bovine viral diarrhoea virus 1 (BVDV-1) and bovine viral diarrhoea virus 2 (BVDV-2). The answer to this question has implication in veterinary diagnostic virology as well as in the BVD control and eradication program in various countries. Phylogenetic analysis of three genetic regions showed three different relationships to both BVDV-1 and BVDV-2 [ 22 ] . To unequivocally solve the relationship, the pestivirus strain Th/04_KhonKaen was recovered from a serum sample of a naturally infected calf and the complete genome sequence was determined [ 39 ] . Phylogenetic analysis of complete genome sequences by Neighborjoining, maximum likelihood, and the Bayesian approach, unanimously placed Th/04_KhonKaen in a single lineage, distinct from the established pestivirus species, and close to BVDV-1 and BVDV-2. A further evolutionary study proposed to term these newly described bovine pestiviruses as BVDV-3 ( Fig. 36.2 ) [ 40 ] .

During the last decades substantial progress has been made in veterinary diagnostic virology at our laboratories at the National Veterinary Institute (SVA) and the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. As the World Organization for Animal Health (OIE) Collaborating Centre for the Biotechnology-Based Diagnosis of Infectious Diseases in Veterinary Medicine , we have developed and tested a wide range of novel molecular diagnostic methods. Many of these novel assays provided powerful novel tools for the improved detection of viruses in veterinary and human medical virology. A wide range of the novel molecular diagnostic methods has been internationally compared in ring tests and validated. In order to illustrate this trend of development, several examples are summarized in this chapter. For molecular methods, upstream nucleic acid extraction is crucial for the success of the downstream diagnostic tests. We came to the conclusion that the real-time PCR principle, using different chemistries, such as TaqMan and PriProET, provides very reliable, highly sensitive and speci fi c novel diagnostic tools for the direct detection of a wide range of pathogens, as it is demonstrated here with several examples. Simultaneously, the real-time PCR technology allows the development of novel DIVA tests , which are highly required for the improved control of infectious diseases, using marker vaccines and accompanied diagnostic packages. In parallel, highthroughput suspension microarray technologies enable the simultaneous detection and identi fi cation of multiple pathogens in single test platforms. The liquid-phase microarray platforms, such as Luminex panels, are accelerating the detection of emerging animal viruses and zoonotic, in particular, the water-and foodborne pathogens. Proximity ligation assay has emerged as a novel method for the highly sensitive and speci fi c detection of the viral proteins. Viral metagenomics and large-scale genome sequencing establish powerful tools for the detection of "unknown" viruses, as well for the identi fi cation of emerging and re-emerging pathogens. These novel approaches strongly support the investigation of disease complexes and/or emerging novel disease scenarios in veterinary diagnostic virology, with regard to diseases in domestic animals and in wildlife, with special regard to zoonotic infections, by following the principles of "One World One Health." 

Validation and quality control of polymerase chain methods used for the diagnosis of infectious diseases. Of fi ce International desEpizooties (OIE), manual of diagnostic tests and vaccines for terrestrial animals (mammals

Advances in viral disease diagnostic and molecular epidemiological techniques

DNA enrichment by functionalized magnetic nanoparticles for onsite and fast detection of virus in biomedical application

Evaluation of automated nucleic acid extraction methods for virus detection in a multiCentre comparative trial

Real time quantitative PCR

Molecular diagnosis of viral diseases, present trends and future aspects: a view from the OIE Collaborating Centre for the application of polymerase chain reaction methods for diagnosis of viral diseases in veterinary medicine

Pan-serotypic detection of foot and mouth disease virus using a minor-groove binder probe reverse transcription polymerase chain reaction assay

Validation of a loop-mediated isothermal ampli fi cation assay for visualised detection of wild-type classical swine fever virus

Development of a loop-mediated isothermal ampli fi cation for visual detection of the HCLV vaccine against classical swine fever in China

A one-step reverse transcriptase loop-mediated isothermal ampli fi cation assay for simple and rapid detection of swine vesicular disease virus

An avirulent chimeric pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus

CP7_E2alf: a safe and ef fi cient marker vaccine strain for oral immunisation of wild boar against classical swine fever virus (CSFV)

Two real-time RT-PCR assays of classical swine fever virus, developed for the genetic differentiation of naturally infected from vaccinated wild boars

Differentiation of C-strain "Riems" or CP7_E2alf vaccinated animals from animals infected by classical swine fever virus fi eld strains using realtime RT-PCR

Escape of classical swine fever C-strain vaccine virus from detection by C-strain speci fi c real-time RT-PCR caused by a point mutation in the primer-binding site

Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus

Development of a novel quantitative real-time PCR assay for the simultaneous detection of all serotypes of foot-andmouth disease virus

Development of a realtime PCR assay based on primer-probe energy transfer for the detection of swine vesicular disease virus

Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus

Evaluation of a primer-probe energy transfer real-time PCR assay for detection of classical swine fever virus

A generic real-time TaqMan assay for speci fi c detection of lapinized Chinese vaccines against classical swine fever

Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species

A TaqMan real-time RT-PCR assay for selective detection of atypical bovine pestiviruses in clinical samples and biological products

Maximum likelihood and Bayesian analyses of a combined nucleotide sequence dataset for genetic characterization of a novel pestivirus, SVA/cont-08

Risk factors for human disease emergence

SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses

Broadly targeted multiprobe QPCR for detection of coronaviruses: coronavirus is common among mallard ducks (Anas platyrhynchos)

Prevalence and phylogenetic relationship of coronaviruses in wild birds from the Bering Strait Area (Beringia)

Universal detection of hepatitis E virus by two real-time PCR assays: TaqMan and Primer-Probe Energy Transfer

Molecular epidemiology of hepatitis E virus in humans, pigs and wild boars in Sweden

Cytokine detection by antibodybased proximity ligation

Novel means of viral antigen identi fi cation: improved detection of avian in fl uenza viruses by proximity ligation

A novel combination of TaqMan RT-PCR and a suspension microarray assay for the detection and species identi fi cation of pestiviruses

A microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies

Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome

Studies of porcine circovirus type 2, porcine boca-like virus and torque teno virus indicate the presence of multiple viral infections in postweaning multisystemic wasting syndrome pigs

Investigations into shaking mink syndrome: an encephalomyelitis of unknown cause in farmed mink (Mustela vison) kits in Scandinavia

Detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics

Virus recovery and full-length sequence analysis of atypical bovine pestivirus Th/04_KhonKaen

Phylogeny, classi fi cation and evolutionary insights into pestiviruses

Acknowledgements This review is summarizing selected recent results of the Joint Research and Development Division in Virology of the National Veterinary Institute (SVA) and the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden, the Collaborating Centre of the OIE. Herewith we thank all our colleagues at SVA and SLU, as well as in the partner laboratories in Sweden and abroad, for their input, for the valuable collaboration and for the interesting results. The work was supported by the Award of Excellence (Excellensbidrag) provided to SB by SLU, by recent EU projects, such as LAB-ON-SITE, CSFV_goDIVA, EPIZONE, ASFRISK, AniBioThreat and by Swedish grants, such as Formas and MSB.