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w viruses in veterinary medicine, detected byetagenomic approaches

ndor Belak a,b,c, Oskar E. Karlsson a,c, Anne-Lie Blomstrom a,c, Mikael Berg a,c,drik Granberg a,c,*

partment of Biomedical Sciences and Veterinary Public Health (BVF), Swedish University of Agricultural Sciences (SLU), Uppsala,

den

partment of Virology, Immunobiology and Parasitology (VIP), National Veterinary Institute (SVA), Uppsala, Sweden

OIE Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (OIE CC), Uppsala, Sweden

tents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

2.1. Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

2.2. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2.3. Amplification of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2.3.1. Multiple displacement amplification (MDA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2.3.2. Sequence-independent single-primer amplification (SISPA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2.4. Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2.4.1. 454/Roche . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2.4.2. Ion Torrent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

T I C L E I N F O

le history:

ived 3 August 2012

ived in revised form 19 January 2013

pted 23 January 2013

ords:

agenomics

encing

nown viruses

s detection

nosis

nown aetiology

A B S T R A C T

In our world, which is faced today with exceptional environmental changes and

dramatically intensifying globalisation, we are encountering challenges due to many new

factors, including the emergence or re-emergence of novel, so far ‘‘unknown’’ infectious

diseases. Although a broad arsenal of diagnostic methods is at our disposal, the majority of

the conventional diagnostic tests is highly virus-specific or is targeted entirely towards a

limited group of infectious agents. This specificity complicates or even hinders the

detection of new or unexpected pathogens, such as new, emerging or re-emerging viruses

or novel viral variants. The recently developed approaches of viral metagenomics provide

an effective novel way to screen samples and detect viruses without previous knowledge

of the infectious agent, thereby enabling a better diagnosis and disease control, in line with

the ‘‘One World, One Health’’ principles (www.oneworldonehealth.org). Using metage-

nomic approaches, we have recently identified a broad variety of new viruses, such as

novel bocaviruses, Torque Teno viruses, astroviruses, rotaviruses and kobuviruses in

porcine disease syndromes, new virus variants in honeybee populations, as well as a range

of other infectious agents in further host species. These findings indicate that the

metagenomic detection of viral pathogens is becoming now a powerful, cultivation-

independent, and useful novel diagnostic tool in veterinary diagnostic virology.

� 2013 Elsevier B.V. All rights reserved.

Corresponding author at: Department of Biomedical Sciences and Veterinary Public Health (BVF), Swedish University of Agricultural Sciences (SLU),

sala, Sweden. Tel.: +46 18 674366.

E-mail address: fredrik.granberg@slu.se (F. Granberg).

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

8-1135/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

://dx.doi.org/10.1016/j.vetmic.2013.01.022

S. Belak et al. / Veterinary Microbiology 165 (2013) 95–10196

2.5. Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2.5.1. Cloud based bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2.5.2. High-performance computing pipeline. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

3.1. Sequence data and bioinformatics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

3.2. Detected viral genomic sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

1. Introduction

Due to intensive globalisation, climatic changes, andviral evolution, among other factors, the emergence ofviruses and new viral diseases has increased in the lastdecades. In this situation, it is crucial to apply powerfulmethods for the broad-range detection and identificationof the emerging viruses. In combination with classicalmethods, the molecular-based techniques provide sensi-tive and rapid means of virus detection and identification(Belak et al., 2009). However, most of the conventionaldiagnostic tests are designed to be virus-specific or aimedat a limited group of infectious agents. This makes themunsuitable for the detection of unexpected and/or com-pletely new viruses, as well as novel viral variants. Incontrast, the novel viral metagenomic approaches allowunbiased detection of a very wide range of infectiousagents in a culture-independent manner and hold thepromise to significantly improve diagnosis and diseasecontrol, in line with the ‘‘One World, One Health’’principles (www.oneworldonehealth.org). Our group atthe OIE CC in Uppsala has established state-of-the artfacilities and practical skills for next-generation sequen-cing (NGS)-based metagenomic detection of pathogens,including ‘‘unknown, new viruses’’ which have a highdegree of divergence from other known infectious agents. Inaddition to the use of established sequencing technologies,such as the 454 sequencing platform (Roche), we arefollowing the ongoing development of further novel high-throughput methodologies, such as the Ion Torrent technol-ogy (Life Technologies). In contrast to earlier sequencing byusing synthesis methods, Ion Torrent relies on a microchip-based array of semiconductor sensors for reading theincorporation of nucleotides onto the synthesis strand,rather than fluorescence signalling (Rothberg et al., 2011).By this way, it opens a new, rapid and more affordable pathin the costly approaches of metagenomics. Herewith webriefly present some technical experiences and summariesof several recent investigations, which were published, orwill be reported in separate specific articles, reporting on theapplication of viral metagenomics to detect ‘‘unknown, newviruses’’ in veterinary medicine.

2. Materials and methods

2.1. Specimens

Samples were collected in various disease syndromegroups in different animal species in three European

metagenomic approaches. The cases were briefly thefollowing (see also Table 1).

Pigs with postweaning multisystemic wasting syndrome

(PMWS): This disease complex is considered to be amultifactorial disorder. Lymph nodes were obtainedfrom 36 animals with PMWS and from 24 individuals,who were not displaying clinical symptoms. Thesamples, collected in Sweden between the years 2003and 2007, originated from animals of 26 herds. Using aninitial metagenomic approach, two of the collectedclinical specimens were investigated for possible viralco-infections. For verification and prevalence estimatesof identified viruses, all samples were analysed.Shaking mink syndrome (SMS): This neurological dis-order affected farmed mink kids in Denmark, asobserved in 2000. Brain homogenates from minksaffected by SMS were used to reproduce the disease inthree healthy individuals. The experimentally infectedanimals developed the disease, however, the appliedconventional methods were unable to detect anyinfectious agent (Gavier-Widen et al., 2004). Thesebrain samples were then subjected to metagenomicanalysis. Specimens from six healthy individuals andthree naturally infected animals were investigatedsimultaneously, to enable confirmation of results.Honeybees with unspecified symptoms: Approximately50 adult worker honeybees, Apis mellifera, weresampled in 2010 from one colony with unusualdepopulation belonging to a commercial apiary of 25hives in the northern part of Spain. A homogenateprepared from 20 whole bees were found to be positivefor Israeli acute paralysis virus (IAPV) by a RT-PCR assay(Palacios et al., 2008). Since IAPV has been linked tocolony collapse disorder (CCD) (Cox-Foster et al., 2007),which is considered to be multifactorial in nature (JoseManuel Sanchez-Vizcaıno, personal communication),we have further investigated these bee samples, byusing metagenomics.Nursery and weaned pigs with diarrhoea: Small intestinesfrom a total of 21 piglets (1–2 weeks old) were collectedat nine different locations in Hungary (Janos Benyedaand Adam Balint, personal communication). Thesamples were examined for viral etiological agents atthe OIE CC in Uppsala, by using a two-step approach.First, all samples were pooled and screened for viralsequences using 454-sequencing. The result was thenused to identify individuals carrying novel viruses byPCR and each of these samples was deep-sequenced

using Ion Torrent technology. countries and analysed at our OIE CC in Uppsala by

Table 1

Summary of published and more recent cases investigated for suspected viral etiological agents at the OIE CC in Uppsala, Sweden.

Syndromes/conditions Sample preparation Amp.

method

Sequencing Data

(Mbp)

Bioinformatics

approach

Identified viral

genomic sequences

References

Pigs with postweaning

multisystemic wasting

syndrome (PMWS)

DNA from nuclease-

treated homogenate of

lymph nodes

MDA 454 GS FLX standard (1/40)a �1.9 Cloud based Porcine circovirus type

2 (PCV-2)

Blomstrom et al. (2009,

2010a)

Torque Teno virus

(TTV)

Porcine boca-like virus

Shaking mink syndrome DNA and RNA from

nuclease-treated

homogenate of brain

tissue

SISPA 454 GS FLX standard (1/8) 10.4 Cloud based Astrovirus (AstV) Blomstrom et al. (2010b)

Honeybees with

unspecified symptoms

DNA and RNA from

nuclease-treated

homogenate of whole

bees

SISPA 454 GS FLX Titanium (1/8) 55.0 High-performance

pipeline

Aphid lethal paralysis

virus (ALPV)

Granberg et al. (2013)

Israeli acute paralysis

virus (IAPV)

Lake Sinai virus (LSV)

Nursery and weaned

pigs with diarrhoea

DNA and RNA from

nuclease-treated

homogenate of small

intestines

SISPA 454 GS FLX Titanium (1/8) 49.3 High-performance

pipeline

Porcine astrovirus

(PAstV)

Karlsson et al.

(manuscript in

preparation)Ion Torrentb 101.7 Kobuvirus

Calicivirus

Rotavirus A

Broilers with severe

interstitial nephritis

DNA and RNA from

nuclease-treated

homogenate of kidneys

SISPA 454 GS FLX Titanium (1/8) 35.5 High-performance

pipeline

Newcastle disease

virus (NDV)

a Fraction of a PicoTiterPlate.b Average amount of sequence data for an Ion 314 chip.

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S. Belak et al. / Veterinary Microbiology 165 (2013) 95–10198

Broilers with severe interstitial nephritis: Severe cases ofinterstitial nephritis in broilers in Hungary made theresponsible veterinarian suspect a new variant ofinfectious bronchitis virus (IBV) in the background.Kidney samples were collected from two affected 10-day-old broilers on a farm (Adam Balint, personalcommunication) and tested by metagenomic analysisat the OIE CC in Uppsala.

2.2. Sample preparation

The sample preparation was performed as earlierdescribed (Blomstrom et al., 2010b). Briefly, solid sampleswere mechanically homogenized and biological fluidswere diluted prior to centrifugation at 4.000 rpm for10 min. The collected supernatants were syringe-filteredthrough disposable 0.45 mm filters (Millipore) and nucle-ase treated with 400 U/ml DNase I (Roche Applied Science)and 8 mg/ml RNase A (Invitrogen) at 37 8C for 2 h. DNAprotected in viral capsids was extracted using the QIAampDNA Mini Kit (Qiagen) and RNA was isolated using TRIzolLS Reagent (Invitrogen) and further purified using RNeasyMini Kit columns (Qiagen).

2.3. Amplification of nucleic acids

2.3.1. Multiple displacement amplification (MDA)

This non-PCR based DNA amplification technique isbased on the use of random primers and Phi29 DNApolymerase to enable double-stranded DNA displacementand primer extension at a constant temperature (Deanet al., 2001). Here the Genomiphi v2 DNA amplification kit(GE healthcare) was used according to the manufacturer’sprotocol.

2.3.2. Sequence-independent single-primer amplification

(SISPA)

After first being introduced (Reyes and Kim, 1991),several variants of sequence-independent single primeramplification (SISPA) have been developed for the purposeof genome sequencing of RNA and DNA viruses. Weemployed a variant of the DNAse-SISPA techniqueintroduced by Allander et al. (2001) and described inour previous publications (Blomstrom et al., 2009, 2010b).

2.4. Sequencing

2.4.1. 454/Roche

The 454/Roche GS FLX system was used in accordancewith established protocols. In brief, amplified nucleic acidwas either directly size-selected by electrophoresis orfragmented to appropriate size (200–600 bp). Librarieswere constructed with multiplex identifier (MID) tags toallow each sample to be run on a fraction (1/8 or less) of aPicoTiterPlate (Roche). While the first runs were per-formed using GS FLX standard sequencing, GS FLXTitanium was used for the later.

2.4.2. Ion Torrent

The Ion Torrent sequencing was performed at theUppsala Genome Center, SciLifeLab, Sweden. In brief,

libraries were built using the Ion Xpress plus fragmentlibrary kit (Life Technologies) followed by size selectionusing the E-Gel CloneWell system (Life Technologies).The quality of generated libraries was assessed using theAgilent Bioanalyzer (Agilent Technologies) with theAgilent High Sensitivity DNA Kit (Agilent Technologies)combined with the Ion library quantification kit qPCR (LifeTechnologies). Each approved library was subjected toemulsion PCR and sequenced on the Ion Torrent PGMsystem using an Ion 314 chip (Life Technologies).

2.5. Bioinformatics

2.5.1. Cloud based bioinformatics

Assembly of sequence reads were performed usingLasergene (DNAstar, Madison, WI) software, homologysearches were performed using the Personal BLASTNavigator software (He et al., 2007), utilizing both BLASTnand BLASTx searchers towards NCBI’s BLAST databases.Based on positive hits, primers were designed to verify thesequence in the sample. When appropriate, full-lengthsequencing was performed using molecular methods tocreate longer amplicons for sequencing, see references(Blomstrom et al., 2009, 2010b).

2.5.2. High-performance computing pipeline

In brief, .fasta and. qual files from extracted SFF fileswere merged into FastQ files using the ‘Combine FASTAand QUAL’ tool. The quality distribution of the reads wasassessed using the FastQC workflow (Andrews, 2012).Data-sets passing the quality threshold were filtered bymapping the sequencing reads towards the known hostgenome (or a close homologue) and then towards knownviruses and bacterial genomes using a short read aligner (Liand Durbin, 2009). Unmapped reads, as well as readsmapped towards known viruses, were subjected toassembly using the Mimicking Intelligent Read Assembly(MIRA) software (Chevreux et al., 1999). Homologysearches with the assembled contigs were divided intothree phases of iteration through BLAST variants towardsthe NCBI nt and nr databases. Final results were thenfiltered for relevant viruses, as well as possible novelviruses, based on protein homology.

The web-based Primer3 software was used to designedprimers according to the obtained consensus sequences forverification of identified viruses by PCR. The sameapproach was also used for gap-filling and primer walkingto obtain near full-length genomes.

3. Results

3.1. Sequence data and bioinformatics

With GS FLX standard sequencing, using one-eighth of aPicoTiterPlate, we initially obtained around 10 Mbp ofsequence data. The introduction of GS FLX Titaniumincreased the output for the same fraction of a plate toaround 30–50 Mbp. For the deep-sequencing in the case ofneonatal pigs with diarrhoea, each run with an Ion Torrent314 chip generated around 100 Mbp. The amounts ofsequence data for the individual cases are listed in Table 1.

Dueseqcanof rviru

3.2.

listrep

4. D

posgen

S. Belak et al. / Veterinary Microbiology 165 (2013) 95–101 99

to differences in starting material, amplification,uencing method and bioinformatic analysis, resultsnot be directly compared between cases. The numberseads and assembled contigs corresponding to specificses have therefore been omitted.

Detected viral genomic sequences

The various viral genomic sequences, identified in theed clinical cases from Sweden, Spain and Hungary areorted in Table 1. The summaries of the findings, briefly:

Pigs with PMWS: In addition to porcine circovirus type 2(PCV-2), a known contributing factor to PMWS, twofurther viruses was detected: Torque Teno virus (TTV)and a novel porcine parvovirus with genetic relation-ship to bocaviruses (Blomstrom et al., 2009). Co-infection by all three viruses was found to be morecommon among PMWS affected animals (71%) ascompared to unaffected pigs (33%), as it is describedin detail in our previous publication (Blomstrom et al.,2010a). These observations indicate a multiple viralinfection in PMWS-affected pigs.Minks with SMS: The analysis revealed the presence of anovel astrovirus (AstV). This virus could also bedetected in naturally infected animals, but not inhealthy minks, using a PCR approach. However, due to asmall sample size, we could not infer any clearassociation between the presence of the AstV in thecentral nervous system (CNS) and the neurologicaldisorder. For further details, see original article(Blomstrom et al., 2010b).Honeybees with unspecified symptoms: Besides IAPV, wecould also identify a variant of aphid lethal paralysisvirus (ALPV) that only recently was recognized to infectbees, and a new strain of Lake Sinai virus (LSV). Inaddition, we also founded that the bees were carriers ofTurnip ringspot virus, an infectious viral agent of plants.For further details, see our joint publication with thegroup of Prof. J.-M. Sanchez-Vizcaıno at UC in Madrid(Granberg et al., 2013).Nursery and weaned pigs with diarrhoea: The deepsequencing of two individual samples indicated thepresence of two new variants of porcine astrovirus. Inaddition, various variants of kobuviruses, calicivirusesand rotavirus A were detected. The results aredescribed in detail by Karlsson et al. (manuscript inpreparation).Broilers with severe interstitial nephritis: The metage-nomic data did not reveal any nucleotide sequencessimilar to IBV. However, the presence of a Newcastledisease virus (NDV) was detected in these clinicalspecimens and the retrieved sequences displayedsignificant homologies (99–100%) to strains propagatedfrom the vaccine strain V4 (manuscript in preparation).

iscussion

The techniques of viral metagenomics open novelsibilities for the direct comparative analysis of theetic compositions of various clinical samples and for

the detection of ‘‘new, emerging viruses.’’ An excellent andvery important example for the practical applicability ofviral metagenomics is the recent detection of Schmallen-berg virus, a novel orthobunyavirus in Europe, with largeepizootiological importance (Hoffmann et al., 2012).Metagenomics have the capacity to detect viruses eitheras single agents or as components in complex infections,such as multifactorial infectious diseases. Considering thiswide range of detection, it is important to remember thatthe discovery of a novel viral genomic sequence by viralmetagenomics does not necessarily prove that thedetected viruses are the causative agents of the studieddiseases or disease complexes. We shall certainly detectnot only a wide range of new or so far undetectedimportant pathogens, but also a high number of non-pathogenic, ubiquitous or omni-present infectious agents.Thus, it is absolutely crucial that the detection of variousviruses it followed by a range of studies, which investigatethe pathogenicity levels and the causative roles of thedetected infectious agents. This means that it is not enoughto report on metagenomic findings, but viral metage-nomics have to be coupled to a range of other comple-mentary investigations, such as attempting the recovery ofthe agent by virus isolation or by transfection and byperforming pathogenicity and epizootiology studies, con-sidering the Koch’s postulates. Simultaneously, the out-come of metagenomics can be further strengthened by arange of associated approaches, such as improved bioin-formatic analysis and deeper sequencing, which mightstrengthen the understanding of the cases and furtherincrease the likelihood to detect low-copy-number virusesin the starting material (Cheval et al., 2011). To increaseour capability to analyse and handle the large datasets,generated by deep sequencing, the original approach to usecommercial software and cloud based bioinformaticssolutions (Blomstrom et al., 2009, 2010b) was replacedby the bioinformatics pipeline described in Section 2 of thisreport. By running the various approaches of metage-nomics, bioinformatics, classical and molecular diagnosticvirology as well as infection biology side-by-side, we shallreach a very advanced new level of pathogen detection,identification, and a more advanced understanding ofinfection biology.

In this short communication, we briefly summarize thepreliminary experiences of the OIE CC in Uppsala, on theestablishment of state-of-the-art metagenomic approachesand platforms, in order to investigate a range of diseasescenarios in various host animal species.

In the case of pigs with PMWS, the metagenomic studyrevealed that in addition to PCV-2, this disease complex isassociated with a novel porcine bocavirus, and with TorqueTeno viruses (TTV). Just like PCV-2 and TTV, the newbocavirus was detected both in PMWS affected andunaffected animals; however, the presence of the viruswas more prevalent among pigs showing the syndromes ofPMWS. It is therefore possible that, similarly to PCV-2,porcine bocaviruses can act as a contributing factor in thecomplex aetiology of PMWS.

The investigation of minks with SMS revealed a novelastrovirus in the CNS possibly associated with theneurological symptoms since only affected animals were

S. Belak et al. / Veterinary Microbiology 165 (2013) 95–101100

found to carry the virus. In agreement with these findings,a recent study reported on an astrovirus as the causativeagent for encephalitis in a 15-year-old boy with agam-maglobulinemia (Quan et al., 2010). Astroviruses aremainly associated with enteric disorders (Moser andSchultz-Cherry, 2005), however, our observations andthe studies of Quan et al. (2010) indicate that it is likelythat these agents are able to be associated even with CNSdiseases in various host species.

The investigations on honeybees, affected by IAPV,showing unclear symptoms, are, to the best of ourknowledge, the first metagenomic study on a honeybeepopulation outside of North America. We believe that thiswork contributes to a global perspective on the honeybeevirome and its diversity. The findings include a variant ofALPV, similar to the recently identified strain Brookings,and a new strain of LSV, a virus demonstrated to be ratherprevalent in the USA but not previously detected in othergeographical areas (Runckel et al., 2011). Co-infection bythese viruses should therefore also be taken into con-sideration when trying to understand the multifactorialdiseases of the honeybees, such as CCD. A furtherinteresting issue is that by detecting a plant virus inhoneybees, we could indicate the role of the bees aspossible vectors of pollen-borne viruses.

The detection of the two new variants of porcineastrovirus in diarrhoea cases of nursery and weaned pigs isalso considered as an important and interesting finding.Since astroviruses are known causative agents of entericdisease complexes in several mammalian species (Moserand Schultz-Cherry, 2005), the findings highlight the roleof pigs as possible reservoirs of viruses that might pose athreat both to humans and other animals. The detectionand identification of additional viruses, i.e., calicivirus androtavirus A indicate a complex aetiology of the studieddiarrhoea cases in swine.

Interestingly, the results of the study on interstitialnephritis in broilers did not support the hypothesis thatthese cases were caused by IBV or associated to this virus.Instead, analysis of the data suggested the association witha variant of NDV similar to the vaccine strain V4, which isin agreement with earlier reports (Nakamura et al., 2008).This is pinpointing the epizootiological situation in thetested poultry populations and opens a path for furthervirological and epidemiological investigations.

This brief summary demonstrates the usefulness ofunbiased metagenomic approaches to investigate theaetiology of various disease scenarios. The studies indicatethat we have obtained the skills and established a state-of-the art platform at the OIE CC in Uppsala for themetagenomic detection of new, previously not knowninfectious agents in various animal species. In the first step, awide range of viruses was detected in several host species, asshown here with several examples. The herewith-summar-ized findings on the detection of various viruses have beenpublished or will be published in specific articles, giving allthe necessary details. We believe that it is extremelyimportant not to stop at this first step, reporting on virusdetection with metagenomics, alias ‘‘virus-hunting,’’ but wehave to continue the work with the next steps, such as trying

development of the diseases. If successful, these steps willopen a new scenario in the diagnosis, control and in theinvestigation of the biological conditions of infectiousdiseases, both in animals and in humans, by following theOne World One Health principles.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

The authors would like to thank Prof. J.M. Sanchez-Vizcaıno, Prof. P. Wallgren, Prof. C. Fossum, Dr. J. Benyeda,Dr. A. Balint and Dr. A.S. Hammer for the collaboration andfor providing interesting samples, Dr. M. Hakhverdyan andDr. M. Leijon for technical support, and the SLU GlobalBioinformatics Centre for assistance with the analysispipeline. Thanks are due to Professors W.I. Lipkin, G.J.Viljoen, M.C. Horzinek and J-F. Valarcher for the supportand for exchanging ideas in metagenomics and inveterinary medical infection-biology. The Award of Excel-lence (Excellensbidrag), provided to SB by the SwedishUniversity of Agricultural Sciences (SLU), created thefacilities for these studies and supported this researchwork all the way.

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