Dataset Information

Investigation of a Canine Parvovirus Outbreak using Next Generation Sequencing.

ABSTRACT: Canine parvovirus (CPV) outbreaks can have a devastating effect in communities with dense dog populations. The interior region of Alaska experienced a CPV outbreak in the winter of 2016 leading to the further investigation of the virus due to reports of increased morbidity and mortality occurring at dog mushing kennels in the area. Twelve rectal-swab specimens from dogs displaying clinical signs consistent with parvoviral-associated disease were processed using next-generation sequencing (NGS) methodologies by targeting RNA transcripts, and therefore detecting only replicating virus. All twelve specimens demonstrated the presence of the CPV transcriptome, with read depths ranging from 2.2X - 12,381X, genome coverage ranging from 44.8-96.5%, and representation of CPV sequencing reads to those of the metagenome background ranging from 0.0015-6.7%. Using the data generated by NGS, the presence of newly evolved, yet known, strains of both CPV-2a and CPV-2b were identified and grouped geographically. Deep-sequencing data provided additional diagnostic information in terms of investigating novel CPV in this outbreak. NGS data in addition to limited serological data provided strong diagnostic evidence that this outbreak most likely arose from unvaccinated or under-vaccinated canines, not from a novel CPV strain incapable of being neutralized by current vaccination efforts.

SUBMITTER: Parker J

PROVIDER: S-EPMC5575238 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Investigation of a Canine Parvovirus Outbreak using Next Generation Sequencing.

Parker Jayme J Murphy Molly M Hueffer Karsten K Chen Jack J

Scientific reports 20170829 1

Canine parvovirus (CPV) outbreaks can have a devastating effect in communities with dense dog populations. The interior region of Alaska experienced a CPV outbreak in the winter of 2016 leading to the further investigation of the virus due to reports of increased morbidity and mortality occurring at dog mushing kennels in the area. Twelve rectal-swab specimens from dogs displaying clinical signs consistent with parvoviral-associated disease were processed using next-generation sequencing (NGS) m ...[more]

PMID: 28852158

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Project description:Accurate normalization of the gene expression assays, using housekeeping genes (HKGs), is critically necessary. To do so, selection of a proper set of HKGs for a specific experiment is of great importance. Despite many studies, there is no consensus about the suitable set of HKGs for implementing in the quantitative real-time PCR analyses of chicken tissues. A limited number of HKGs have been widely used. However, wide utilization of a little number of HKGs for all tissues is challenging. The emergence of high-throughput gene expression RNA-seq data has enabled the simultaneous comparison of the stability of multiple HKGs. Therefore, employing the average coefficient of variations of at least three datasets per tissue, we sorted all reliably expressed genes (REGs; with FPKM ≥ 1 in at least one sample) and introduced the top 10 most suitable and stable reference genes for each of the 16 chicken tissues. We evaluated the consistency of the results of five tissues using the same methodology on other datasets. Furthermore, we assessed 96 previously widely used HKGs (WU-HKGs) in order to challenge the accuracy of the previous studies. The New Tuxedo software suite was used for the main analyses. The results revealed novel, different sets of reference genes for each of the tissues with 17 common genes among the top 10 genes lists of 16 tissues. The results did disprove the suitability of WU-HKGs such as Actb, Ldha, Scd, B2m, and Hprt1 for any of the tissues examined. On the contrary, a total of 6, 13, 14, 23, and 32 validated housekeeping genes (V-HKGs) were discovered as the most stable and suitable reference genes for muscle, spleen, liver, heart, and kidney tissues, respectively. Although we identified a few new HKGs usable for multiple tissues, the selection of suitable HKGs is required to be tissue specific. The newly introduced reference genes from the present study, despite lacking experimental validation, will be able to contribute to the more accurate normalization for future expression analysis of chicken genes.

Project description:Parvovirus B19 (B19V) is a ssDNA human virus, responsible for an ample range of clinical manifestations. Sequencing of B19V DNA from clinical samples is frequently reported in the literature to assign genotype (genotypes 1-3) and for finer molecular epidemiological tracing. The increasing availability of Next Generation Sequencing (NGS) with its depth of coverage potentially yields information on intrinsic sequence heterogeneity; however, integration of this information in analysis of sequence variation is not routinely obtained. The present work investigated genomic sequence heterogeneity within and between B19V isolates by application of NGS techniques, and by the development of a novel dedicated bioinformatic tool and analysis pipeline, yielding information on two newly defined parameters. The first, α-diversity, is a measure of the amount and distribution of position-specific, normalised Shannon Entropy, as a measure of intra-sample sequence heterogeneity. The second, σ-diversity, is a measure of the amount of inter-sample sequence heterogeneity, also incorporating information on α-diversity. Based on these indexes, further cluster analysis can be performed. A set of 24 high-titre viraemic samples was investigated. Of these, 23 samples were genotype 1 and one sample was genotype 2. Genotype 1 isolates showed low α-diversity values, with only a few samples showing distinct position-specific polymorphisms; a few genetically related clusters emerged when analysing inter-sample distances, correlated to the year of isolation; the single genotype 2 isolate showed the highest α-diversity, even if not presenting polymorphisms, and was an evident outlier when analysing inter-sample distance. In conclusion, NGS analysis and the bioinformatic tool and pipeline developed and used in the present work can be considered effective tools for investigating sequence diversity, an observable parameter that can be incorporated into the quasispecies theory framework to yield a better insight into viral evolution dynamics.

Dataset Information

Investigation of a Canine Parvovirus Outbreak using Next Generation Sequencing.

Publications

Investigation of a Canine Parvovirus Outbreak using Next Generation Sequencing.

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