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New viruses in veterinary medicine, detected by metagenomic approaches
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VETMIC-6085; No. of Pages 7 Veterinary Microbiology xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Review
New viruses in veterinary medicine, detected by metagenomic approaches Sa´ndor Bela´k a,b,c, Oskar E. Karlsson a,c, Anne-Lie Blomstro¨m a,c, Mikael Berg a,c, Fredrik Granberg a,c,* a Department of Biomedical Sciences and Veterinary Public Health (BVF), Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden b Department of Virology, Immunobiology and Parasitology (VIP), National Veterinary Institute (SVA), Uppsala, Sweden c The OIE Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (OIE CC), Uppsala, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 August 2012 Received in revised form 19 January 2013 Accepted 23 January 2013
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 metagenomic 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, cultivationindependent, and useful novel diagnostic tool in veterinary diagnostic virology. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Metagenomics Sequencing Unknown viruses Virus detection Diagnosis Unknown aetiology
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. 2.3. Amplification of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple displacement amplification (MDA) . . . . . . . . . . . . 2.3.1. Sequence-independent single-primer amplification (SISPA) 2.3.2. Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. 454/Roche . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Ion Torrent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2.
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* Corresponding author at: Department of Biomedical Sciences and Veterinary Public Health (BVF), Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden. Tel.: +46 18 674366. E-mail address: [email protected] (F. Granberg). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.01.022
Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
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2.5.
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Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . Cloud based bioinformatics . . . . . . . . . . 2.5.1. 2.5.2. High-performance computing pipeline. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequence data and bioinformatics. . . . . . . . . . . . 3.1. Detected viral genomic sequences. . . . . . . . . . . . 3.2. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Due to intensive globalisation, climatic changes, and viral evolution, among other factors, the emergence of viruses and new viral diseases has increased in the last decades. In this situation, it is crucial to apply powerful methods for the broad-range detection and identification of the emerging viruses. In combination with classical methods, the molecular-based techniques provide sensitive and rapid means of virus detection and identification (Bela´k et al., 2009). However, most of the conventional diagnostic tests are designed to be virus-specific or aimed at a limited group of infectious agents. This makes them unsuitable for the detection of unexpected and/or completely new viruses, as well as novel viral variants. In contrast, the novel viral metagenomic approaches allow unbiased detection of a very wide range of infectious agents in a culture-independent manner and hold the promise to significantly improve diagnosis and disease control, in line with the ‘‘One World, One Health’’ principles (www.oneworldonehealth.org). Our group at the OIE CC in Uppsala has established state-of-the art facilities and practical skills for next-generation sequencing (NGS)-based metagenomic detection of pathogens, including ‘‘unknown, new viruses’’ which have a high degree of divergence from other known infectious agents. In addition to the use of established sequencing technologies, such as the 454 sequencing platform (Roche), we are following the ongoing development of further novel highthroughput methodologies, such as the Ion Torrent technology (Life Technologies). In contrast to earlier sequencing by using synthesis methods, Ion Torrent relies on a microchipbased array of semiconductor sensors for reading the incorporation 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 path in the costly approaches of metagenomics. Herewith we briefly present some technical experiences and summaries of several recent investigations, which were published, or will be reported in separate specific articles, reporting on the application of viral metagenomics to detect ‘‘unknown, new viruses’’ in veterinary medicine. 2. Materials and methods 2.1. Specimens Samples were collected in various disease syndrome groups in different animal species in three European countries and analysed at our OIE CC in Uppsala by
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metagenomic approaches. The cases were briefly the following (see also Table 1). Pigs with postweaning multisystemic wasting syndrome (PMWS): This disease complex is considered to be a multifactorial disorder. Lymph nodes were obtained from 36 animals with PMWS and from 24 individuals, who were not displaying clinical symptoms. The samples, collected in Sweden between the years 2003 and 2007, originated from animals of 26 herds. Using an initial metagenomic approach, two of the collected clinical specimens were investigated for possible viral co-infections. For verification and prevalence estimates of identified viruses, all samples were analysed. Shaking mink syndrome (SMS): This neurological disorder affected farmed mink kids in Denmark, as observed in 2000. Brain homogenates from minks affected by SMS were used to reproduce the disease in three healthy individuals. The experimentally infected animals developed the disease, however, the applied conventional methods were unable to detect any infectious agent (Gavier-Wide´n et al., 2004). These brain samples were then subjected to metagenomic analysis. Specimens from six healthy individuals and three naturally infected animals were investigated simultaneously, to enable confirmation of results. Honeybees with unspecified symptoms: Approximately 50 adult worker honeybees, Apis mellifera, were sampled in 2010 from one colony with unusual depopulation belonging to a commercial apiary of 25 hives in the northern part of Spain. A homogenate prepared from 20 whole bees were found to be positive for Israeli acute paralysis virus (IAPV) by a RT-PCR assay (Palacios et al., 2008). Since IAPV has been linked to colony collapse disorder (CCD) (Cox-Foster et al., 2007), which is considered to be multifactorial in nature (Jose´ Manuel Sa´nchez-Vizcaı´no, personal communication), we have further investigated these bee samples, by using metagenomics. Nursery and weaned pigs with diarrhoea: Small intestines from a total of 21 piglets (1–2 weeks old) were collected at nine different locations in Hungary (Ja´nos Benyeda and A´da´m Ba´lint, personal communication). The samples were examined for viral etiological agents at the OIE CC in Uppsala, by using a two-step approach. First, all samples were pooled and screened for viral sequences using 454-sequencing. The result was then used to identify individuals carrying novel viruses by PCR and each of these samples was deep-sequenced using Ion Torrent technology.
Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
Sample preparation
Amp. method
Sequencing
Pigs with postweaning multisystemic wasting syndrome (PMWS)
DNA from nucleasetreated homogenate of lymph nodes
MDA
454 GS FLX standard (1/40)a
Shaking mink syndrome
DNA and RNA from nuclease-treated homogenate of brain tissue
SISPA
Honeybees with unspecified symptoms
DNA and RNA from nuclease-treated homogenate of whole bees
Nursery and weaned pigs with diarrhoea
Broilers with severe interstitial nephritis a b
Bioinformatics approach
Identified viral genomic sequences
References
1.9
Cloud based
Porcine circovirus type 2 (PCV-2) Torque Teno virus (TTV) Porcine boca-like virus
Blomstro¨m et al. (2009, 2010a)
454 GS FLX standard (1/8)
10.4
Cloud based
Astrovirus (AstV)
Blomstro¨m et al. (2010b)
SISPA
454 GS FLX Titanium (1/8)
55.0
High-performance pipeline
Aphid lethal paralysis virus (ALPV) Israeli acute paralysis virus (IAPV) Lake Sinai virus (LSV)
Granberg et al. (2013)
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) Kobuvirus Calicivirus Rotavirus A
Karlsson et al. (manuscript in preparation)
DNA and RNA from nuclease-treated homogenate of kidneys
SISPA
High-performance pipeline
Newcastle disease virus (NDV)
Ion Torrentb
454 GS FLX Titanium (1/8)
Data (Mbp)
101.7
35.5
Fraction of a PicoTiterPlate. Average amount of sequence data for an Ion 314 chip.
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Syndromes/conditions
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Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
Table 1 Summary of published and more recent cases investigated for suspected viral etiological agents at the OIE CC in Uppsala, Sweden.
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Broilers with severe interstitial nephritis: Severe cases of interstitial nephritis in broilers in Hungary made the responsible veterinarian suspect a new variant of infectious bronchitis virus (IBV) in the background. Kidney samples were collected from two affected 10day-old broilers on a farm (A´da´m Ba´lint, personal communication) and tested by metagenomic analysis at the OIE CC in Uppsala. 2.2. Sample preparation The sample preparation was performed as earlier described (Blomstro¨m et al., 2010b). Briefly, solid samples were mechanically homogenized and biological fluids were diluted prior to centrifugation at 4.000 rpm for 10 min. The collected supernatants were syringe-filtered through disposable 0.45 mm filters (Millipore) and nuclease treated with 400 U/ml DNase I (Roche Applied Science) and 8 mg/ml RNase A (Invitrogen) at 37 8C for 2 h. DNA protected in viral capsids was extracted using the QIAamp DNA Mini Kit (Qiagen) and RNA was isolated using TRIzol LS Reagent (Invitrogen) and further purified using RNeasy Mini Kit columns (Qiagen).
libraries were built using the Ion Xpress plus fragment library kit (Life Technologies) followed by size selection using the E-Gel CloneWell system (Life Technologies). The quality of generated libraries was assessed using the Agilent Bioanalyzer (Agilent Technologies) with the Agilent High Sensitivity DNA Kit (Agilent Technologies) combined with the Ion library quantification kit qPCR (Life Technologies). Each approved library was subjected to emulsion PCR and sequenced on the Ion Torrent PGM system using an Ion 314 chip (Life Technologies). 2.5. Bioinformatics 2.5.1. Cloud based bioinformatics Assembly of sequence reads were performed using Lasergene (DNAstar, Madison, WI) software, homology searches were performed using the Personal BLAST Navigator software (He et al., 2007), utilizing both BLASTn and BLASTx searchers towards NCBI’s BLAST databases. Based on positive hits, primers were designed to verify the sequence in the sample. When appropriate, full-length sequencing was performed using molecular methods to create longer amplicons for sequencing, see references (Blomstro¨m et al., 2009, 2010b).
2.3. Amplification of nucleic acids 2.3.1. Multiple displacement amplification (MDA) This non-PCR based DNA amplification technique is based on the use of random primers and Phi29 DNA polymerase to enable double-stranded DNA displacement and primer extension at a constant temperature (Dean et al., 2001). Here the Genomiphi v2 DNA amplification kit (GE healthcare) was used according to the manufacturer’s protocol. 2.3.2. Sequence-independent single-primer amplification (SISPA) After first being introduced (Reyes and Kim, 1991), several variants of sequence-independent single primer amplification (SISPA) have been developed for the purpose of genome sequencing of RNA and DNA viruses. We employed a variant of the DNAse-SISPA technique introduced by Allander et al. (2001) and described in our previous publications (Blomstro¨m et al., 2009, 2010b). 2.4. Sequencing 2.4.1. 454/Roche The 454/Roche GS FLX system was used in accordance with established protocols. In brief, amplified nucleic acid was either directly size-selected by electrophoresis or fragmented to appropriate size (200–600 bp). Libraries were constructed with multiplex identifier (MID) tags to allow each sample to be run on a fraction (1/8 or less) of a PicoTiterPlate (Roche). While the first runs were performed using GS FLX standard sequencing, GS FLX Titanium was used for the later. 2.4.2. Ion Torrent The Ion Torrent sequencing was performed at the Uppsala Genome Center, SciLifeLab, Sweden. In brief,
2.5.2. High-performance computing pipeline In brief, .fasta and. qual files from extracted SFF files were merged into FastQ files using the ‘Combine FASTA and QUAL’ tool. The quality distribution of the reads was assessed using the FastQC workflow (Andrews, 2012). Data-sets passing the quality threshold were filtered by mapping the sequencing reads towards the known host genome (or a close homologue) and then towards known viruses and bacterial genomes using a short read aligner (Li and Durbin, 2009). Unmapped reads, as well as reads mapped towards known viruses, were subjected to assembly using the Mimicking Intelligent Read Assembly (MIRA) software (Chevreux et al., 1999). Homology searches with the assembled contigs were divided into three phases of iteration through BLAST variants towards the NCBI nt and nr databases. Final results were then filtered for relevant viruses, as well as possible novel viruses, based on protein homology. The web-based Primer3 software was used to designed primers according to the obtained consensus sequences for verification of identified viruses by PCR. The same approach was also used for gap-filling and primer walking to obtain near full-length genomes. 3. Results 3.1. Sequence data and bioinformatics With GS FLX standard sequencing, using one-eighth of a PicoTiterPlate, we initially obtained around 10 Mbp of sequence data. The introduction of GS FLX Titanium increased the output for the same fraction of a plate to around 30–50 Mbp. For the deep-sequencing in the case of neonatal pigs with diarrhoea, each run with an Ion Torrent 314 chip generated around 100 Mbp. The amounts of sequence data for the individual cases are listed in Table 1.
Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
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Due to differences in starting material, amplification, sequencing method and bioinformatic analysis, results cannot be directly compared between cases. The numbers of reads and assembled contigs corresponding to specific viruses have therefore been omitted. 3.2. Detected viral genomic sequences The various viral genomic sequences, identified in the listed clinical cases from Sweden, Spain and Hungary are reported 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, two further viruses was detected: Torque Teno virus (TTV) and a novel porcine parvovirus with genetic relationship to bocaviruses (Blomstro¨m et al., 2009). Coinfection by all three viruses was found to be more common among PMWS affected animals (71%) as compared to unaffected pigs (33%), as it is described in detail in our previous publication (Blomstro¨m et al., 2010a). These observations indicate a multiple viral infection in PMWS-affected pigs. Minks with SMS: The analysis revealed the presence of a novel astrovirus (AstV). This virus could also be detected in naturally infected animals, but not in healthy minks, using a PCR approach. However, due to a small sample size, we could not infer any clear association between the presence of the AstV in the central nervous system (CNS) and the neurological disorder. For further details, see original article (Blomstro¨m et al., 2010b). Honeybees with unspecified symptoms: Besides IAPV, we could also identify a variant of aphid lethal paralysis virus (ALPV) that only recently was recognized to infect bees, and a new strain of Lake Sinai virus (LSV). In addition, we also founded that the bees were carriers of Turnip ringspot virus, an infectious viral agent of plants. For further details, see our joint publication with the group of Prof. J.-M. Sa´nchez-Vizcaı´no at UC in Madrid (Granberg et al., 2013). Nursery and weaned pigs with diarrhoea: The deep sequencing of two individual samples indicated the presence of two new variants of porcine astrovirus. In addition, various variants of kobuviruses, caliciviruses and rotavirus A were detected. The results are described in detail by Karlsson et al. (manuscript in preparation). Broilers with severe interstitial nephritis: The metagenomic data did not reveal any nucleotide sequences similar to IBV. However, the presence of a Newcastle disease virus (NDV) was detected in these clinical specimens and the retrieved sequences displayed significant homologies (99–100%) to strains propagated from the vaccine strain V4 (manuscript in preparation). 4. Discussion The techniques of viral metagenomics open novel possibilities for the direct comparative analysis of the genetic compositions of various clinical samples and for
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the detection of ‘‘new, emerging viruses.’’ An excellent and very important example for the practical applicability of viral metagenomics is the recent detection of Schmallenberg virus, a novel orthobunyavirus in Europe, with large epizootiological importance (Hoffmann et al., 2012). Metagenomics have the capacity to detect viruses either as single agents or as components in complex infections, such as multifactorial infectious diseases. Considering this wide range of detection, it is important to remember that the discovery of a novel viral genomic sequence by viral metagenomics does not necessarily prove that the detected viruses are the causative agents of the studied diseases or disease complexes. We shall certainly detect not only a wide range of new or so far undetected important pathogens, but also a high number of nonpathogenic, ubiquitous or omni-present infectious agents. Thus, it is absolutely crucial that the detection of various viruses it followed by a range of studies, which investigate the pathogenicity levels and the causative roles of the detected infectious agents. This means that it is not enough to report on metagenomic findings, but viral metagenomics have to be coupled to a range of other complementary investigations, such as attempting the recovery of the agent by virus isolation or by transfection and by performing pathogenicity and epizootiology studies, considering the Koch’s postulates. Simultaneously, the outcome of metagenomics can be further strengthened by a range of associated approaches, such as improved bioinformatic analysis and deeper sequencing, which might strengthen the understanding of the cases and further increase the likelihood to detect low-copy-number viruses in the starting material (Cheval et al., 2011). To increase our capability to analyse and handle the large datasets, generated by deep sequencing, the original approach to use commercial software and cloud based bioinformatics solutions (Blomstro¨m et al., 2009, 2010b) was replaced by the bioinformatics pipeline described in Section 2 of this report. By running the various approaches of metagenomics, bioinformatics, classical and molecular diagnostic virology as well as infection biology side-by-side, we shall reach a very advanced new level of pathogen detection, identification, and a more advanced understanding of infection biology. In this short communication, we briefly summarize the preliminary experiences of the OIE CC in Uppsala, on the establishment of state-of-the-art metagenomic approaches and platforms, in order to investigate a range of disease scenarios in various host animal species. In the case of pigs with PMWS, the metagenomic study revealed that in addition to PCV-2, this disease complex is associated with a novel porcine bocavirus, and with Torque Teno viruses (TTV). Just like PCV-2 and TTV, the new bocavirus was detected both in PMWS affected and unaffected animals; however, the presence of the virus was more prevalent among pigs showing the syndromes of PMWS. It is therefore possible that, similarly to PCV-2, porcine bocaviruses can act as a contributing factor in the complex aetiology of PMWS. The investigation of minks with SMS revealed a novel astrovirus in the CNS possibly associated with the neurological symptoms since only affected animals were
Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
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found to carry the virus. In agreement with these findings, a recent study reported on an astrovirus as the causative agent for encephalitis in a 15-year-old boy with agammaglobulinemia (Quan et al., 2010). Astroviruses are mainly associated with enteric disorders (Moser and Schultz-Cherry, 2005), however, our observations and the studies of Quan et al. (2010) indicate that it is likely that these agents are able to be associated even with CNS diseases in various host species. The investigations on honeybees, affected by IAPV, showing unclear symptoms, are, to the best of our knowledge, the first metagenomic study on a honeybee population outside of North America. We believe that this work contributes to a global perspective on the honeybee virome and its diversity. The findings include a variant of ALPV, similar to the recently identified strain Brookings, and a new strain of LSV, a virus demonstrated to be rather prevalent in the USA but not previously detected in other geographical areas (Runckel et al., 2011). Co-infection by these viruses should therefore also be taken into consideration when trying to understand the multifactorial diseases of the honeybees, such as CCD. A further interesting issue is that by detecting a plant virus in honeybees, we could indicate the role of the bees as possible vectors of pollen-borne viruses. The detection of the two new variants of porcine astrovirus in diarrhoea cases of nursery and weaned pigs is also considered as an important and interesting finding. Since astroviruses are known causative agents of enteric disease complexes in several mammalian species (Moser and Schultz-Cherry, 2005), the findings highlight the role of pigs as possible reservoirs of viruses that might pose a threat both to humans and other animals. The detection and identification of additional viruses, i.e., calicivirus and rotavirus A indicate a complex aetiology of the studied diarrhoea cases in swine. Interestingly, the results of the study on interstitial nephritis in broilers did not support the hypothesis that these cases were caused by IBV or associated to this virus. Instead, analysis of the data suggested the association with a variant of NDV similar to the vaccine strain V4, which is in agreement with earlier reports (Nakamura et al., 2008). This is pinpointing the epizootiological situation in the tested poultry populations and opens a path for further virological and epidemiological investigations. This brief summary demonstrates the usefulness of unbiased metagenomic approaches to investigate the aetiology of various disease scenarios. The studies indicate that we have obtained the skills and established a state-ofthe art platform at the OIE CC in Uppsala for the metagenomic detection of new, previously not known infectious agents in various animal species. In the first step, a wide range of viruses was detected in several host species, as shown here with several examples. The herewith-summarized findings on the detection of various viruses have been published or will be published in specific articles, giving all the necessary details. We believe that it is extremely important not to stop at this first step, reporting on virus detection with metagenomics, alias ‘‘virus-hunting,’’ but we have to continue the work with the next steps, such as trying to isolate the viral agents and to study their role in the
development of the diseases. If successful, these steps will open a new scenario in the diagnosis, control and in the investigation of the biological conditions of infectious diseases, both in animals and in humans, by following the One World One Health principles. Conflict of interest The authors declare no conflict of interest. Acknowledgments The authors would like to thank Prof. J.M. Sa´nchezVizcaı´no, Prof. P. Wallgren, Prof. C. Fossum, Dr. J. Benyeda, Dr. A´. Ba´lint and Dr. A.S. Hammer for the collaboration and for providing interesting samples, Dr. M. Hakhverdyan and Dr. M. Leijon for technical support, and the SLU Global Bioinformatics Centre for assistance with the analysis pipeline. Thanks are due to Professors W.I. Lipkin, G.J. Viljoen, M.C. Horzinek and J-F. Valarcher for the support and for exchanging ideas in metagenomics and in veterinary medical infection-biology. The Award of Excellence (Excellensbidrag), provided to SB by the Swedish University of Agricultural Sciences (SLU), created the facilities for these studies and supported this research work all the way. References Allander, T., Emerson, S.U., Engle, R.E., Purcell, R.H., Bukh, J., 2001. A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species. Proc. Natl. Acad. Sci. U.S.A. 98, 11609–11614. Andrews, S., 2012. FASTQC – a quality control tool for high throughput sequence data. Babraham Bioinformatics. Bela´k, S., Thoren, P., LeBlanc, N., Viljoen, G., 2009. Advances in viral disease diagnostic and molecular epidemiological technologies. Expert Rev. Mol. Diagn. 9, 367–381. Blomstro¨m, A.L., Bela´k, S., Fossum, C., Fuxler, L., Wallgren, P., Berg, M., 2010a. 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. Virus Res. 152, 59–64. Blomstro¨m, A.L., Bela´k, S., Fossum, C., McKillen, J., Allan, G., Wallgren, P., Berg, M., 2009. Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome. Virus Res. 146, 125–129. Blomstro¨m, A.L., Wide´n, F., Hammer, A.S., Bela´k, S., Berg, M., 2010b. Detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics. J. Clin. Microbiol. 48, 4392–4396. Cheval, J., Sauvage, V., Frangeul, L., Dacheux, L., Guigon, G., Dumey, N., Pariente, K., Rousseaux, C., Dorange, F., Berthet, N., Brisse, S., Moszer, I., Bourhy, H., Manuguerra, C.J., Lecuit, M., Burguiere, A., Caro, V., Eloit, M., 2011. Evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples. J. Clin. Microbiol. 49, 3268–3275. Chevreux, B., Wetter, T., Suhai, S., 1999. Genome sequence assembly using trace signals and additional sequence information. In: Computer Science and Biology: Proceedings of the German Conference on Bioinformatics (GCB). pp. 45–56. Cox-Foster, D.L., Conlan, S., Holmes, E.C., Palacios, G., Evans, J.D., Moran, N.A., Quan, P.L., Briese, T., Hornig, M., Geiser, D.M., Martinson, V., vanEngelsdorp, D., Kalkstein, A.L., Drysdale, A., Hui, J., Zhai, J., Cui, L., Hutchison, S.K., Simons, J.F., Egholm, M., Pettis, J.S., Lipkin, W.I., 2007. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318, 283–287. Dean, F.B., Nelson, J.R., Giesler, T.L., Lasken, R.S., 2001. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099.
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VETMIC-6085; No. of Pages 7 S. Bela´k et al. / Veterinary Microbiology xxx (2013) xxx–xxx Gavier-Wide´n, D., Brojer, C., Dietz, H.H., Englund, L., Hammer, A.S., Hedlund, K.O., af Segerstad, C.H., Nilsson, K., Nowotny, N., Puurula, V., Thoren, P., Uhlhorn, H., Weissenbock, H., Agren, E., Klingeborn, B., 2004. Investigations into shaking mink syndrome: an encephalomyelitis of unknown cause in farmed mink (Mustela vison) kits in Scandinavia. J. Vet. Diagn. Invest. 16, 305–312. Granberg, F., Vicente-Rubiano, M., Rubio-Guerri, C., Karlsson, O.E., Kukielka, D., Bela´k, S., Sa´nchez-Vizcaı´no, J.M., 2013. Metagenomic detection of viral pathogens in Spanish honeybees: co-infection by aphid lethal paralysis, Israel acute paralysis and Lake Sinai viruses. PLoS ONE, in press. He, J., Dai, X., Zhao, X., 2007. PLAN: a web platform for automating highthroughput BLAST searches and for managing and mining results. BMC Bioinformatics 8, 53. Hoffmann, B., Scheuch, M., Hoper, D., Jungblut, R., Holsteg, M., Schirrmeier, H., Eschbaumer, M., Goller, K.V., Wernike, K., Fischer, M., Breithaupt, A., Mettenleiter, T.C., Beer, M., 2012. Novel orthobunyavirus in Cattle, Europe, 2011. Emerg. Infect. Dis. 18, 469–472. Li, H., Durbin, R., 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760. Moser, L.A., Schultz-Cherry, S., 2005. Pathogenesis of astrovirus infection. Viral Immunol. 18, 4–10. Nakamura, K., Ohtsu, N., Nakamura, T., Yamamoto, Y., Yamada, M., Mase, M., Imai, K., 2008. Pathologic and immunohistochemical studies of Newcastle disease (ND) in broiler chickens vaccinated with ND: severe nonpurulent encephalitis and necrotizing pancreatitis. Vet. Pathol. 45, 928–933.
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Palacios, G., Hui, J., Quan, P.L., Kalkstein, A., Honkavuori, K.S., Bussetti, A.V., Conlan, S., Evans, J., Chen, Y.P., vanEngelsdorp, D., Efrat, H., Pettis, J., Cox-Foster, D., Holmes, E.C., Briese, T., Lipkin, W.I., 2008. Genetic analysis of Israel acute paralysis virus: distinct clusters are circulating in the United States. J. Virol. 82, 6209–6217. Quan, P.L., Wagner, T.A., Briese, T., Torgerson, T.R., Hornig, M., Tashmukhamedova, A., Firth, C., Palacios, G., Baisre-De-Leon, A., Paddock, C.D., Hutchison, S.K., Egholm, M., Zaki, S.R., Goldman, J.E., Ochs, H.D., Lipkin, W.I., 2010. Astrovirus encephalitis in boy with X-linked agammaglobulinemia. Emerg. Infect. Dis. 16, 918–925. Reyes, G.R., Kim, J.P., 1991. Sequence-independent, single-primer amplification (SISPA) of complex DNA populations. Mol. Cell. Probes 5, 473– 481. Rothberg, J.M., Hinz, W., Rearick, T.M., Schultz, J., Mileski, W., Davey, M., Leamon, J.H., Johnson, K., Milgrew, M.J., Edwards, M., Hoon, J., Simons, J.F., Marran, D., Myers, J.W., Davidson, J.F., Branting, A., Nobile, J.R., Puc, B.P., Light, D., Clark, T.A., Huber, M., Branciforte, J.T., Stoner, I.B., Cawley, S.E., Lyons, M., Fu, Y., Homer, N., Sedova, M., Miao, X., Reed, B., Sabina, J., Feierstein, E., Schorn, M., Alanjary, M., Dimalanta, E., Dressman, D., Kasinskas, R., Sokolsky, T., Fidanza, J.A., Namsaraev, E., McKernan, K.J., Williams, A., Roth, G.T., Bustillo, J., 2011. An integrated semiconductor device enabling non-optical genome sequencing. Nature 475, 348–352. Runckel, C., Flenniken, M.L., Engel, J.C., Ruby, J.G., Ganem, D., Andino, R., DeRisi, J.L., 2011. Temporal analysis of the honey bee microbiome reveals four novel viruses and seasonal prevalence of known viruses, Nosema, and Crithidia. PLoS One 6, e20656.
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VETMIC-6085; No. of Pages 7 Veterinary Microbiology xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Review
New viruses in veterinary medicine, detected by metagenomic approaches Sa´ndor Bela´k a,b,c, Oskar E. Karlsson a,c, Anne-Lie Blomstro¨m a,c, Mikael Berg a,c, Fredrik Granberg a,c,* a Department of Biomedical Sciences and Veterinary Public Health (BVF), Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden b Department of Virology, Immunobiology and Parasitology (VIP), National Veterinary Institute (SVA), Uppsala, Sweden c The OIE Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (OIE CC), Uppsala, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 August 2012 Received in revised form 19 January 2013 Accepted 23 January 2013
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 metagenomic 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, cultivationindependent, and useful novel diagnostic tool in veterinary diagnostic virology. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Metagenomics Sequencing Unknown viruses Virus detection Diagnosis Unknown aetiology
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. 2.3. Amplification of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple displacement amplification (MDA) . . . . . . . . . . . . 2.3.1. Sequence-independent single-primer amplification (SISPA) 2.3.2. Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. 454/Roche . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Ion Torrent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2.
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* Corresponding author at: Department of Biomedical Sciences and Veterinary Public Health (BVF), Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden. Tel.: +46 18 674366. E-mail address: [email protected] (F. Granberg). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.01.022
Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
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2.5.
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Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . Cloud based bioinformatics . . . . . . . . . . 2.5.1. 2.5.2. High-performance computing pipeline. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequence data and bioinformatics. . . . . . . . . . . . 3.1. Detected viral genomic sequences. . . . . . . . . . . . 3.2. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Due to intensive globalisation, climatic changes, and viral evolution, among other factors, the emergence of viruses and new viral diseases has increased in the last decades. In this situation, it is crucial to apply powerful methods for the broad-range detection and identification of the emerging viruses. In combination with classical methods, the molecular-based techniques provide sensitive and rapid means of virus detection and identification (Bela´k et al., 2009). However, most of the conventional diagnostic tests are designed to be virus-specific or aimed at a limited group of infectious agents. This makes them unsuitable for the detection of unexpected and/or completely new viruses, as well as novel viral variants. In contrast, the novel viral metagenomic approaches allow unbiased detection of a very wide range of infectious agents in a culture-independent manner and hold the promise to significantly improve diagnosis and disease control, in line with the ‘‘One World, One Health’’ principles (www.oneworldonehealth.org). Our group at the OIE CC in Uppsala has established state-of-the art facilities and practical skills for next-generation sequencing (NGS)-based metagenomic detection of pathogens, including ‘‘unknown, new viruses’’ which have a high degree of divergence from other known infectious agents. In addition to the use of established sequencing technologies, such as the 454 sequencing platform (Roche), we are following the ongoing development of further novel highthroughput methodologies, such as the Ion Torrent technology (Life Technologies). In contrast to earlier sequencing by using synthesis methods, Ion Torrent relies on a microchipbased array of semiconductor sensors for reading the incorporation 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 path in the costly approaches of metagenomics. Herewith we briefly present some technical experiences and summaries of several recent investigations, which were published, or will be reported in separate specific articles, reporting on the application of viral metagenomics to detect ‘‘unknown, new viruses’’ in veterinary medicine. 2. Materials and methods 2.1. Specimens Samples were collected in various disease syndrome groups in different animal species in three European countries and analysed at our OIE CC in Uppsala by
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metagenomic approaches. The cases were briefly the following (see also Table 1). Pigs with postweaning multisystemic wasting syndrome (PMWS): This disease complex is considered to be a multifactorial disorder. Lymph nodes were obtained from 36 animals with PMWS and from 24 individuals, who were not displaying clinical symptoms. The samples, collected in Sweden between the years 2003 and 2007, originated from animals of 26 herds. Using an initial metagenomic approach, two of the collected clinical specimens were investigated for possible viral co-infections. For verification and prevalence estimates of identified viruses, all samples were analysed. Shaking mink syndrome (SMS): This neurological disorder affected farmed mink kids in Denmark, as observed in 2000. Brain homogenates from minks affected by SMS were used to reproduce the disease in three healthy individuals. The experimentally infected animals developed the disease, however, the applied conventional methods were unable to detect any infectious agent (Gavier-Wide´n et al., 2004). These brain samples were then subjected to metagenomic analysis. Specimens from six healthy individuals and three naturally infected animals were investigated simultaneously, to enable confirmation of results. Honeybees with unspecified symptoms: Approximately 50 adult worker honeybees, Apis mellifera, were sampled in 2010 from one colony with unusual depopulation belonging to a commercial apiary of 25 hives in the northern part of Spain. A homogenate prepared from 20 whole bees were found to be positive for Israeli acute paralysis virus (IAPV) by a RT-PCR assay (Palacios et al., 2008). Since IAPV has been linked to colony collapse disorder (CCD) (Cox-Foster et al., 2007), which is considered to be multifactorial in nature (Jose´ Manuel Sa´nchez-Vizcaı´no, personal communication), we have further investigated these bee samples, by using metagenomics. Nursery and weaned pigs with diarrhoea: Small intestines from a total of 21 piglets (1–2 weeks old) were collected at nine different locations in Hungary (Ja´nos Benyeda and A´da´m Ba´lint, personal communication). The samples were examined for viral etiological agents at the OIE CC in Uppsala, by using a two-step approach. First, all samples were pooled and screened for viral sequences using 454-sequencing. The result was then used to identify individuals carrying novel viruses by PCR and each of these samples was deep-sequenced using Ion Torrent technology.
Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
Sample preparation
Amp. method
Sequencing
Pigs with postweaning multisystemic wasting syndrome (PMWS)
DNA from nucleasetreated homogenate of lymph nodes
MDA
454 GS FLX standard (1/40)a
Shaking mink syndrome
DNA and RNA from nuclease-treated homogenate of brain tissue
SISPA
Honeybees with unspecified symptoms
DNA and RNA from nuclease-treated homogenate of whole bees
Nursery and weaned pigs with diarrhoea
Broilers with severe interstitial nephritis a b
Bioinformatics approach
Identified viral genomic sequences
References
1.9
Cloud based
Porcine circovirus type 2 (PCV-2) Torque Teno virus (TTV) Porcine boca-like virus
Blomstro¨m et al. (2009, 2010a)
454 GS FLX standard (1/8)
10.4
Cloud based
Astrovirus (AstV)
Blomstro¨m et al. (2010b)
SISPA
454 GS FLX Titanium (1/8)
55.0
High-performance pipeline
Aphid lethal paralysis virus (ALPV) Israeli acute paralysis virus (IAPV) Lake Sinai virus (LSV)
Granberg et al. (2013)
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) Kobuvirus Calicivirus Rotavirus A
Karlsson et al. (manuscript in preparation)
DNA and RNA from nuclease-treated homogenate of kidneys
SISPA
High-performance pipeline
Newcastle disease virus (NDV)
Ion Torrentb
454 GS FLX Titanium (1/8)
Data (Mbp)
101.7
35.5
Fraction of a PicoTiterPlate. Average amount of sequence data for an Ion 314 chip.
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Syndromes/conditions
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Table 1 Summary of published and more recent cases investigated for suspected viral etiological agents at the OIE CC in Uppsala, Sweden.
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Broilers with severe interstitial nephritis: Severe cases of interstitial nephritis in broilers in Hungary made the responsible veterinarian suspect a new variant of infectious bronchitis virus (IBV) in the background. Kidney samples were collected from two affected 10day-old broilers on a farm (A´da´m Ba´lint, personal communication) and tested by metagenomic analysis at the OIE CC in Uppsala. 2.2. Sample preparation The sample preparation was performed as earlier described (Blomstro¨m et al., 2010b). Briefly, solid samples were mechanically homogenized and biological fluids were diluted prior to centrifugation at 4.000 rpm for 10 min. The collected supernatants were syringe-filtered through disposable 0.45 mm filters (Millipore) and nuclease treated with 400 U/ml DNase I (Roche Applied Science) and 8 mg/ml RNase A (Invitrogen) at 37 8C for 2 h. DNA protected in viral capsids was extracted using the QIAamp DNA Mini Kit (Qiagen) and RNA was isolated using TRIzol LS Reagent (Invitrogen) and further purified using RNeasy Mini Kit columns (Qiagen).
libraries were built using the Ion Xpress plus fragment library kit (Life Technologies) followed by size selection using the E-Gel CloneWell system (Life Technologies). The quality of generated libraries was assessed using the Agilent Bioanalyzer (Agilent Technologies) with the Agilent High Sensitivity DNA Kit (Agilent Technologies) combined with the Ion library quantification kit qPCR (Life Technologies). Each approved library was subjected to emulsion PCR and sequenced on the Ion Torrent PGM system using an Ion 314 chip (Life Technologies). 2.5. Bioinformatics 2.5.1. Cloud based bioinformatics Assembly of sequence reads were performed using Lasergene (DNAstar, Madison, WI) software, homology searches were performed using the Personal BLAST Navigator software (He et al., 2007), utilizing both BLASTn and BLASTx searchers towards NCBI’s BLAST databases. Based on positive hits, primers were designed to verify the sequence in the sample. When appropriate, full-length sequencing was performed using molecular methods to create longer amplicons for sequencing, see references (Blomstro¨m et al., 2009, 2010b).
2.3. Amplification of nucleic acids 2.3.1. Multiple displacement amplification (MDA) This non-PCR based DNA amplification technique is based on the use of random primers and Phi29 DNA polymerase to enable double-stranded DNA displacement and primer extension at a constant temperature (Dean et al., 2001). Here the Genomiphi v2 DNA amplification kit (GE healthcare) was used according to the manufacturer’s protocol. 2.3.2. Sequence-independent single-primer amplification (SISPA) After first being introduced (Reyes and Kim, 1991), several variants of sequence-independent single primer amplification (SISPA) have been developed for the purpose of genome sequencing of RNA and DNA viruses. We employed a variant of the DNAse-SISPA technique introduced by Allander et al. (2001) and described in our previous publications (Blomstro¨m et al., 2009, 2010b). 2.4. Sequencing 2.4.1. 454/Roche The 454/Roche GS FLX system was used in accordance with established protocols. In brief, amplified nucleic acid was either directly size-selected by electrophoresis or fragmented to appropriate size (200–600 bp). Libraries were constructed with multiplex identifier (MID) tags to allow each sample to be run on a fraction (1/8 or less) of a PicoTiterPlate (Roche). While the first runs were performed using GS FLX standard sequencing, GS FLX Titanium was used for the later. 2.4.2. Ion Torrent The Ion Torrent sequencing was performed at the Uppsala Genome Center, SciLifeLab, Sweden. In brief,
2.5.2. High-performance computing pipeline In brief, .fasta and. qual files from extracted SFF files were merged into FastQ files using the ‘Combine FASTA and QUAL’ tool. The quality distribution of the reads was assessed using the FastQC workflow (Andrews, 2012). Data-sets passing the quality threshold were filtered by mapping the sequencing reads towards the known host genome (or a close homologue) and then towards known viruses and bacterial genomes using a short read aligner (Li and Durbin, 2009). Unmapped reads, as well as reads mapped towards known viruses, were subjected to assembly using the Mimicking Intelligent Read Assembly (MIRA) software (Chevreux et al., 1999). Homology searches with the assembled contigs were divided into three phases of iteration through BLAST variants towards the NCBI nt and nr databases. Final results were then filtered for relevant viruses, as well as possible novel viruses, based on protein homology. The web-based Primer3 software was used to designed primers according to the obtained consensus sequences for verification of identified viruses by PCR. The same approach was also used for gap-filling and primer walking to obtain near full-length genomes. 3. Results 3.1. Sequence data and bioinformatics With GS FLX standard sequencing, using one-eighth of a PicoTiterPlate, we initially obtained around 10 Mbp of sequence data. The introduction of GS FLX Titanium increased the output for the same fraction of a plate to around 30–50 Mbp. For the deep-sequencing in the case of neonatal pigs with diarrhoea, each run with an Ion Torrent 314 chip generated around 100 Mbp. The amounts of sequence data for the individual cases are listed in Table 1.
Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
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Due to differences in starting material, amplification, sequencing method and bioinformatic analysis, results cannot be directly compared between cases. The numbers of reads and assembled contigs corresponding to specific viruses have therefore been omitted. 3.2. Detected viral genomic sequences The various viral genomic sequences, identified in the listed clinical cases from Sweden, Spain and Hungary are reported 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, two further viruses was detected: Torque Teno virus (TTV) and a novel porcine parvovirus with genetic relationship to bocaviruses (Blomstro¨m et al., 2009). Coinfection by all three viruses was found to be more common among PMWS affected animals (71%) as compared to unaffected pigs (33%), as it is described in detail in our previous publication (Blomstro¨m et al., 2010a). These observations indicate a multiple viral infection in PMWS-affected pigs. Minks with SMS: The analysis revealed the presence of a novel astrovirus (AstV). This virus could also be detected in naturally infected animals, but not in healthy minks, using a PCR approach. However, due to a small sample size, we could not infer any clear association between the presence of the AstV in the central nervous system (CNS) and the neurological disorder. For further details, see original article (Blomstro¨m et al., 2010b). Honeybees with unspecified symptoms: Besides IAPV, we could also identify a variant of aphid lethal paralysis virus (ALPV) that only recently was recognized to infect bees, and a new strain of Lake Sinai virus (LSV). In addition, we also founded that the bees were carriers of Turnip ringspot virus, an infectious viral agent of plants. For further details, see our joint publication with the group of Prof. J.-M. Sa´nchez-Vizcaı´no at UC in Madrid (Granberg et al., 2013). Nursery and weaned pigs with diarrhoea: The deep sequencing of two individual samples indicated the presence of two new variants of porcine astrovirus. In addition, various variants of kobuviruses, caliciviruses and rotavirus A were detected. The results are described in detail by Karlsson et al. (manuscript in preparation). Broilers with severe interstitial nephritis: The metagenomic data did not reveal any nucleotide sequences similar to IBV. However, the presence of a Newcastle disease virus (NDV) was detected in these clinical specimens and the retrieved sequences displayed significant homologies (99–100%) to strains propagated from the vaccine strain V4 (manuscript in preparation). 4. Discussion The techniques of viral metagenomics open novel possibilities for the direct comparative analysis of the genetic compositions of various clinical samples and for
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the detection of ‘‘new, emerging viruses.’’ An excellent and very important example for the practical applicability of viral metagenomics is the recent detection of Schmallenberg virus, a novel orthobunyavirus in Europe, with large epizootiological importance (Hoffmann et al., 2012). Metagenomics have the capacity to detect viruses either as single agents or as components in complex infections, such as multifactorial infectious diseases. Considering this wide range of detection, it is important to remember that the discovery of a novel viral genomic sequence by viral metagenomics does not necessarily prove that the detected viruses are the causative agents of the studied diseases or disease complexes. We shall certainly detect not only a wide range of new or so far undetected important pathogens, but also a high number of nonpathogenic, ubiquitous or omni-present infectious agents. Thus, it is absolutely crucial that the detection of various viruses it followed by a range of studies, which investigate the pathogenicity levels and the causative roles of the detected infectious agents. This means that it is not enough to report on metagenomic findings, but viral metagenomics have to be coupled to a range of other complementary investigations, such as attempting the recovery of the agent by virus isolation or by transfection and by performing pathogenicity and epizootiology studies, considering the Koch’s postulates. Simultaneously, the outcome of metagenomics can be further strengthened by a range of associated approaches, such as improved bioinformatic analysis and deeper sequencing, which might strengthen the understanding of the cases and further increase the likelihood to detect low-copy-number viruses in the starting material (Cheval et al., 2011). To increase our capability to analyse and handle the large datasets, generated by deep sequencing, the original approach to use commercial software and cloud based bioinformatics solutions (Blomstro¨m et al., 2009, 2010b) was replaced by the bioinformatics pipeline described in Section 2 of this report. By running the various approaches of metagenomics, bioinformatics, classical and molecular diagnostic virology as well as infection biology side-by-side, we shall reach a very advanced new level of pathogen detection, identification, and a more advanced understanding of infection biology. In this short communication, we briefly summarize the preliminary experiences of the OIE CC in Uppsala, on the establishment of state-of-the-art metagenomic approaches and platforms, in order to investigate a range of disease scenarios in various host animal species. In the case of pigs with PMWS, the metagenomic study revealed that in addition to PCV-2, this disease complex is associated with a novel porcine bocavirus, and with Torque Teno viruses (TTV). Just like PCV-2 and TTV, the new bocavirus was detected both in PMWS affected and unaffected animals; however, the presence of the virus was more prevalent among pigs showing the syndromes of PMWS. It is therefore possible that, similarly to PCV-2, porcine bocaviruses can act as a contributing factor in the complex aetiology of PMWS. The investigation of minks with SMS revealed a novel astrovirus in the CNS possibly associated with the neurological symptoms since only affected animals were
Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022
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found to carry the virus. In agreement with these findings, a recent study reported on an astrovirus as the causative agent for encephalitis in a 15-year-old boy with agammaglobulinemia (Quan et al., 2010). Astroviruses are mainly associated with enteric disorders (Moser and Schultz-Cherry, 2005), however, our observations and the studies of Quan et al. (2010) indicate that it is likely that these agents are able to be associated even with CNS diseases in various host species. The investigations on honeybees, affected by IAPV, showing unclear symptoms, are, to the best of our knowledge, the first metagenomic study on a honeybee population outside of North America. We believe that this work contributes to a global perspective on the honeybee virome and its diversity. The findings include a variant of ALPV, similar to the recently identified strain Brookings, and a new strain of LSV, a virus demonstrated to be rather prevalent in the USA but not previously detected in other geographical areas (Runckel et al., 2011). Co-infection by these viruses should therefore also be taken into consideration when trying to understand the multifactorial diseases of the honeybees, such as CCD. A further interesting issue is that by detecting a plant virus in honeybees, we could indicate the role of the bees as possible vectors of pollen-borne viruses. The detection of the two new variants of porcine astrovirus in diarrhoea cases of nursery and weaned pigs is also considered as an important and interesting finding. Since astroviruses are known causative agents of enteric disease complexes in several mammalian species (Moser and Schultz-Cherry, 2005), the findings highlight the role of pigs as possible reservoirs of viruses that might pose a threat both to humans and other animals. The detection and identification of additional viruses, i.e., calicivirus and rotavirus A indicate a complex aetiology of the studied diarrhoea cases in swine. Interestingly, the results of the study on interstitial nephritis in broilers did not support the hypothesis that these cases were caused by IBV or associated to this virus. Instead, analysis of the data suggested the association with a variant of NDV similar to the vaccine strain V4, which is in agreement with earlier reports (Nakamura et al., 2008). This is pinpointing the epizootiological situation in the tested poultry populations and opens a path for further virological and epidemiological investigations. This brief summary demonstrates the usefulness of unbiased metagenomic approaches to investigate the aetiology of various disease scenarios. The studies indicate that we have obtained the skills and established a state-ofthe art platform at the OIE CC in Uppsala for the metagenomic detection of new, previously not known infectious agents in various animal species. In the first step, a wide range of viruses was detected in several host species, as shown here with several examples. The herewith-summarized findings on the detection of various viruses have been published or will be published in specific articles, giving all the necessary details. We believe that it is extremely important not to stop at this first step, reporting on virus detection with metagenomics, alias ‘‘virus-hunting,’’ but we have to continue the work with the next steps, such as trying to isolate the viral agents and to study their role in the
development of the diseases. If successful, these steps will open a new scenario in the diagnosis, control and in the investigation of the biological conditions of infectious diseases, both in animals and in humans, by following the One World One Health principles. Conflict of interest The authors declare no conflict of interest. Acknowledgments The authors would like to thank Prof. J.M. Sa´nchezVizcaı´no, Prof. P. Wallgren, Prof. C. Fossum, Dr. J. Benyeda, Dr. A´. Ba´lint and Dr. A.S. Hammer for the collaboration and for providing interesting samples, Dr. M. Hakhverdyan and Dr. M. Leijon for technical support, and the SLU Global Bioinformatics Centre for assistance with the analysis pipeline. Thanks are due to Professors W.I. Lipkin, G.J. Viljoen, M.C. Horzinek and J-F. Valarcher for the support and for exchanging ideas in metagenomics and in veterinary medical infection-biology. The Award of Excellence (Excellensbidrag), provided to SB by the Swedish University of Agricultural Sciences (SLU), created the facilities for these studies and supported this research work all the way. References Allander, T., Emerson, S.U., Engle, R.E., Purcell, R.H., Bukh, J., 2001. A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species. Proc. Natl. Acad. Sci. U.S.A. 98, 11609–11614. Andrews, S., 2012. FASTQC – a quality control tool for high throughput sequence data. Babraham Bioinformatics. Bela´k, S., Thoren, P., LeBlanc, N., Viljoen, G., 2009. Advances in viral disease diagnostic and molecular epidemiological technologies. Expert Rev. Mol. Diagn. 9, 367–381. Blomstro¨m, A.L., Bela´k, S., Fossum, C., Fuxler, L., Wallgren, P., Berg, M., 2010a. 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. Virus Res. 152, 59–64. Blomstro¨m, A.L., Bela´k, S., Fossum, C., McKillen, J., Allan, G., Wallgren, P., Berg, M., 2009. Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome. Virus Res. 146, 125–129. Blomstro¨m, A.L., Wide´n, F., Hammer, A.S., Bela´k, S., Berg, M., 2010b. Detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics. J. Clin. Microbiol. 48, 4392–4396. Cheval, J., Sauvage, V., Frangeul, L., Dacheux, L., Guigon, G., Dumey, N., Pariente, K., Rousseaux, C., Dorange, F., Berthet, N., Brisse, S., Moszer, I., Bourhy, H., Manuguerra, C.J., Lecuit, M., Burguiere, A., Caro, V., Eloit, M., 2011. Evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples. J. Clin. Microbiol. 49, 3268–3275. Chevreux, B., Wetter, T., Suhai, S., 1999. Genome sequence assembly using trace signals and additional sequence information. In: Computer Science and Biology: Proceedings of the German Conference on Bioinformatics (GCB). pp. 45–56. Cox-Foster, D.L., Conlan, S., Holmes, E.C., Palacios, G., Evans, J.D., Moran, N.A., Quan, P.L., Briese, T., Hornig, M., Geiser, D.M., Martinson, V., vanEngelsdorp, D., Kalkstein, A.L., Drysdale, A., Hui, J., Zhai, J., Cui, L., Hutchison, S.K., Simons, J.F., Egholm, M., Pettis, J.S., Lipkin, W.I., 2007. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318, 283–287. Dean, F.B., Nelson, J.R., Giesler, T.L., Lasken, R.S., 2001. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099.
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Please cite this article in press as: Bela´k, S., et al., New viruses in veterinary medicine, detected by metagenomic approaches. Vet. Microbiol. (2013), http://dx.doi.org/10.1016/j.vetmic.2013.01.022