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Virus Detection Using Metagenomic Sequencing of Swine Nasal and Rectal Swabs Ben M

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Virus Detection Using Metagenomic Sequencing of Swine Nasal and Rectal Swabs Ben M Diagnostic notes Peer reviewed Virus detection using metagenomic sequencing of swine nasal and rectal swabs Ben M. Hause, MS, PhD; Joshua W. Duff, DVM; Alan Scheidt, DVM; Gary Anderson, DVM, PhD Summary novo and analyzed by BLASTN to identify and Senecavirus A. Other significant swine Advances in DNA sequencing have in- viruses present in the samples. Twenty seven viruses detected less frequently include creased our ability to generate large amounts different viruses were identified. Reads porcine circovirus type 2, porcine epidemic of sequence data at lower costs. These devel- similar to a diverse family of single-stranded diarrhea virus, and porcine deltacoronavirus. opments have enabled microbial detection circular DNA viruses were identified in Together, these results suggest that metage- and characterization directly from clinical nearly every sample (47 of 50). Other viruses nomic sequencing is a powerful tool for specimens, known as metagenomic sequenc- identified at all five sampling sites and in virus detection and characterization. over half of the samples were bocavirus, toro- ing. Viral metagenomic sequencing was Keywords: swine, DNA sequencing, virus, virus, posavirus, torque teno virus, IAS virus, performed on five nasal- and five fecal-swab Senecavirus A, virome pools collected from each of two primary and picobirnavirus, and teschovirus. Viruses two secondary market slaughterhouses and a identified in multiple sites in greater than Received: December 30, 2015 cull-swine buying station in the southeastern 20% of the samples included enterovirus, Accepted: April 15, 2016 United States. Sequences were assembled de parvovirus, influenza A virus, sapelovirus, Resumen - Detección de virus utilizando en los cinco sitios de muestreo y en más de la ont permis la détection microbienne et la secuenciación metagenómica de hisopos mitad de las muestras fueron bocavirus, toro- caractérisation directement à partir de spéci- nasales y rectales porcinos virus, posavirus, virus torque teno, virus IAS, mens cliniques, connus sous l’appellation de picobirnavirus, y teschovirus. Los virus identi- séquençage métagénomique. Le séquençage Los avances en la secuenciación de DNA ficados en sitios múltiples en más del 20% de métagénomique viral a été effectué sur cinq han incrementado nuestra habilidad para las muestras incluyeron enterovirus, parvovi- pools d’écouvillons nasaux et cinq pools generar grandes cantidades de información rus, virus de la influenza A, sapelovirus, y Sen- d’écouvillons rectaux prélevés de chacun de secuencias a costos más bajos. Estos de- ecavirus A. Otros virus porcinos significativos de deux abattoirs primaires et deux abat- sarrollos han permitido la caracterización detectados con menor frecuencia incluyeron toirs secondaires, ainsi que d’une station y la detección microbiana directamente circovirus porcino tipo 2, virus de la diarrea d’achat d’animaux réformés dans le sud-est de especímenes clínicos, conocida como epidémica porcina, y el deltacoronavirus por- des États-Unis. Les séquences ont été as- secuenciación metagenómica. Se realizó la cino. Conjuntamente, estos resultados sugie- semblées de novo et analysées par BLASTN secuenciación metagenómica viral en cinco ren que la secuenciación metagenómica es una afin d’identifier les virus présents dans les grupos de cinco muestras nasales y fecales herramienta poderosa para la caracterización échantillons. Vingt-sept virus différents ont recolectadas de dos mataderos primarios y y detección de virus. été identifiés. Des lectures similaires à une dos secundarios y de una estación de compra famille variée de virus à ADN circulaire de animales de desecho en el sureste de los simple brin ont été identifiées dans presque Estados Unidos. Las secuencias se mon- Résumé - Détection de virus en utilisant le tous les échantillons (47 des 50). Les autres taron de novo y se analizaron por medio de séquençage métagénomique d’écouvillons virus identifiés dans tous les sites échantil- BLASTN para identificar los virus presentes nasaux et rectaux lonnés et dans plus de la moitié des échantil- en las muestras. Se identificaron veintisiete lons étaient des bocavirus, des torovirus, des virus diferentes. Se identificaron lecturas Les avancées dans le séquençage de l’ADN posavirus, des torque teno virus, les virus semejantes a una familia diversa de virus de ont augmenté la capacité à générer de IAS, les picobirnavirus, et les teschovirus. DNA circular de cadena simple casi en cada grandes quantités de données de séquences Les virus identifiés dans des sites multiples muestra (47 de 50). Otros virus identificados à des coûts moindres. Ces développements dans plus de 20% des échantillons incluaient les enterovirus, les parvorirus, le virus de BMH, GA: Kansas State Veterinary Diagnostic Laboratory, Manhattan, Kansas. l’influenza A, les sapelovirus, et le Senecavi- rus A. Les autres virus porcins significatifs JWD: Maxwell Foods, Inc, Goldsboro, North Carolina. détectés moins fréquemment incluaient le AS: Boehringer Ingelheim Vetmedica, Inc, St Joseph, Missouri. circovirus porcin de type 2, le virus de la Dr Hause is currently with Cambridge Technologies, 1525 Bioscience Drive, Worthington, MN 56187; diarrhée épidémique porcine, et le deltacoro- Tel: 507-220-3916; E-mail: [email protected]. navirus porcin. Ces résultats suggèrent que le séquençage métagénomique est un outil http://www.aasv.org/shap.html This article is available online at . puissant pour la détection et la caractérisa- Hause BM, Duff JW, Scheidt A, et al. Virus detection using metagenomic sequencing of swine nasal tion des virus. and rectal swabs. J Swine Health Prod. 2016;24(6):304–308. 304 Journal of Swine Health and Production — November and December 2016 2016_Nov-Dec_Book 1.indb 304 9/30/2016 1:40:22 PM or greater than a decade, DNA Large numbers of viruses are known to Amplicons were quantified using a Qubit sequencing has been widely used by infect swine, and new viruses are routinely fluorometer (ThermoFisher Scientific), and swine veterinarians to study the epide- being discovered. While several publications libraries were prepared by the standard Fmiology of clinically important viruses such have explored the swine virome, we have Nextera XT Library Preparation Kit as porcine reproductive and respiratory syn- limited understanding of the clinical signifi- (Illumina, San Diego, California) protocol. drome virus (PRRSV) and influenza A virus cance of most of these viruses.7,8 The goal of All 50 libraries were pooled and sequenced (IAV). Traditional DNA sequencing meth- this study was to characterize the swine vi- using paired 300-bp reads on a single Miseq odology, developed by Sanger, has the ad- rome at points of animal concentration and (Illumina) run. Sequence reads were parsed vantages of low cost, fast turnaround times, commingling. using barcodes incorporated during library and relatively long read lengths.1 Com- preparation and imported into CLC Ge- monly, the gene encoding the most variable Sampling protocol nomics Workbench (CLC Bio, Waltham, immunodominant viral protein is targeted. Nasal and fecal swabs were collected in uni- Massachusetts). Reads were mapped to Polymerase chain reaction (PCR) is used to versal viral transport medium from five in- the host genome (Sus scrofa), and from the amplify the gene of interest, and the purified dividual pigs derived from a single producer unmapped reads, contigs were assembled PCR product is subjected to Sanger se- and assembled into nasal- and fecal-swab de novo. Contigs, the consensus sequences quencing using a DNA primer that binds to pools. Pigs from five producers were collect- derived from overlapping DNA sequences, the PCR product. Multiple sequencing reads ed per site. Five sites in total were sampled were analyzed by the basic local alignment are utilized to achieve complete gene cover- in August 2015. Samples were collected by search tool nucleotides (BLASTN). Contigs age. Prices charged by veterinary diagnostic a veterinarian, and all animals were clini- with expectation (E) values (a measure of -10 were laboratories in the United States vary, but cally healthy. Sites 1 and 2 were abattoirs database hit strength) less than 10 considered positive for virus identification. are typically approximately $100 to $180 per that purchased top-quality hogs (primary For viruses with multiple BLASTN hits to sample, with results available within days. A market). Sites 3 and 4 were cull-swine abat- similar viral species, virus identity was as- significant disadvantage of Sanger sequenc- toirs. Site 5 was a cull-swine buying station. signed to a higher, more inclusive taxonomic ing is a requirement for a priori knowledge Animals from sites 1 to 4 were greater than level, typically the genus. of an organism’s presence in a sample, as well 20 weeks of age, while animals at Site 5 were as sufficient sequence homology to enable greater than 10 weeks of age. binding of the sequencing primer. Results New DNA sequencing technologies have Metagenomic sequencing Viruses detected in primary market swabs become more commonplace in diagnostic Metagenomic sequencing was performed are shown in Figure 1. A total of 19 dif- 2,3 laboratories. Next-generation sequencing approximately as previously described.5 ferent viruses were detected. Contigs with platforms generate massive amounts of se- Swabs in transport medium were vortexed, significantE values to bocavirus, torovirus, quencing information at low cost. Owing to and pools of five nasal or fecal
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